In presence of this striking analogy, it is unnecessary to insist

further on the details of the acquired immunity of animals against the

experimental disease set up by the micro-organism of typhoid fever. It

will be better to select another example from the group of bacilli. Let

us first take the acquired immunity against the bacillus of blue pus

(_Bacillus pyocyaneus_) which for many years has been regarded as the

best example in which to study this kind of immunity. Charrin, who was

the first to obtain disease with this bacillus experimentally, published

several notes[333] on the acquired immunity of the rabbit against it. He

demonstrated the possibility of vaccinating this animal not only with

living bacilli, but also with the products of their culture; he studied

the blood serum of vaccinated animals, comparing it with the serum of

normal rabbits, especially as to its action on the development of the

_Bacillus pyocyaneus_. Although unable to find any bactericidal power

properly so called in the serum of immunised rabbits, Charrin was the

first to draw attention to certain modifications undergone by the

bacilli when grown in this medium. He noted that under these conditions

no pyocyanin was produced, and, in collaboration with Roger, he

demonstrated that, in the serum of the vaccinated rabbit, the Bacillus

pyocyaneus forms packets composed of little chains of greater or less

length, whilst in the serum of the normal, susceptible rabbit, it

develops in the form of normal rods, the rods for the most part being

isolated.

[Sidenote: [245]]

From his experiments _in vitro_ Charrin concluded that there was marked

enfeeblement of the functions of the _Bacillus pyocyaneus_ when

submitted to the action of the vaccinated animal organism. Bouchard[334]

has gone so far as to develop a theory of acquired immunity, in which

the principal part is attributed to the impossibility of the

micro-organism, after it has invaded the refractory animal, secreting

its fluid products; there is no vascular dilatation and diapedesis does

not take place. A comparative observation of the phenomena observed in

rabbits that are susceptible to the pyocyanic disease and of those met

with in vaccinated rabbits, most clearly, however, demonstrates the

impossibility of accepting Bouchard’s interpretation. The inoculation of

the bacillus of blue pus below the skin of the ear of the normal

(unvaccinated) rabbit sets up extensive inflammatory reaction with

marked hyperaemia; the diapedesis of the white corpuscles takes place at

a comparatively late stage of the process and phagocytosis is neither

set up nor completed until very late. On the other hand, in vaccinated

rabbits, infected in the same way, the hyperaemia of the ear is

insignificant, but diapedesis occurs very early and phagocytosis

commences at once. It is not, therefore, the impossibility for the

leucocytes to traverse the vessel wall, owing to the absence of the

dilatation of the veins, which prevents them from making their way

rapidly to the field of battle; it is their imperfect positive

sensitiveness that is accountable for the tardy and incomplete

phagocytosis. This interpretation is confirmed in other cases of

acquired immunity.

[Sidenote: [246]]

More recently, Paul Müller[335] has laid special stress on the part

played by the bactericidal action of the serum of animals that have been

vaccinated against the pyocyanic disease. For him the negative results

obtained by his predecessors lose their significance, since all their

experiments were carried out under conditions of aërobiosis, whilst it

is only in the absence of free oxygen that this bactericidal power can

be exerted at all freely. Müller, therefore, set himself to compare

under anaerobic conditions the bactericidal action on the _Bacillus

pyocyaneus_ of serums coming from normal and from vaccinated animals. He

succeeded in demonstrating that the blood serum of vaccinated animals is

more bactericidal than that of normal rabbits. Before, however, drawing

any conclusion from this observation, the following question must be

answered: Are the phenomena observed _in vitro_ comparable with those

seen in the living animal? In preceding chapters it has been shown so

often that the blood serum obtained after the separation of the

extravascular clot, can in no way be identified with the plasma of the

circulating blood, that it is unnecessary to argue this matter further.

If we wish to gain a clear idea of the mechanism of immunity in the

living animal we must observe the course of events in the vaccinated

animal and not draw conclusions from observations _in vitro_ except

after strict examination. All the works on pyocyanic immunity above

summarised lie under the reproach that in them this rule has not been

adhered to.

Since the discovery of Pfeiffer’s phenomenon in animals that have been

vaccinated against the cholera vibrio, much greater care has been taken

to attend to the changes that occur in the animal that enjoys acquired

immunity. Wassermann[336] was the first to attempt to apply Pfeiffer’s

discovery to the _Bacillus pyocyaneus_. With a race of this bacillus

rendered more virulent he succeeded in producing a fatal experimental

malady in the guinea-pig against which he was able by various methods to

vaccinate these animals.

[Sidenote: [247]]

He thus describes the phenomena observed in the peritoneal cavity of

immunised guinea-pigs. Soon after injection the bacilli of blue pus

become motionless, then “the rods swell up and melt, like wax in hot

water. The formation of granules, such as occur in the cholera vibrio,

has been observed but rarely. The process recalls rather that which

takes place in experimental typhoid fever, as described by R. Pfeiffer.

In all cases the phenomenon of solution takes place entirely in the

fluid of the exudation, without any co-operation on the part of the

leucocytes” (p. 284). We see that we have still to do with a kind of

attenuated Pfeiffer’s phenomenon, without any granular change, but with

an immobilisation of the bacilli. As Wassermann has remained satisfied

with the examination of the peritoneal content which, as we know, gives

but an imperfect picture of acquired immunity, Gheorghiewsky[337] set

himself to study the question more thoroughly under my direction. With

this object he vaccinated a series of guinea-pigs with living bacilli of

blue pus, a sure method of obtaining acquired immunity. On examining the

peritoneal fluid (withdrawn shortly after the injection of the bacilli)

of the vaccinated guinea-pigs, he found that the bacilli were motionless

and had undergone a certain degree of agglutination. They were not

transformed into granules but became thicker and somewhat more dumpy.

These changes are observed during the period of phagolysis, when only a

few scattered leucocytes are to be found in the fluid of the peritoneal

cavity. About two hours after the injection of the bacilli the

leucocytes begin to reappear in the peritoneal exudation, more

especially the microphages, which lose no time in seizing the bacilli,

some of which become transformed into granules. A few hours later the

exudation, containing a multitude of leucocytes, no longer contains any

free bacilli: all are found inside the microphages. Nevertheless, if a

drop of the exudation now be withdrawn and kept for some time at a

temperature of 37° C., it will be found that the bacilli multiply inside

the dead phagocytes outside the animal. We thus obtain colonies of

bacilli, a fact which clearly proves that these bacilli whilst still

alive have been ingested by the leucocytes. This experiment is,

therefore, very similar to the one we have described in connection with

Gamaleia’s vibrio.

[Sidenote: [248]]

Even at a later period, 24 or 30 hours after the injection of the

bacilli, that is to say at a period when an examination of the exudation

no longer reveals the presence of bacilli, the sowing of a drop of this

exudation on a nutrient medium still gives isolated colonies of the

_Bacillus pyocyaneus_ capable of producing the characteristic pigments.

At a still later period, when the peritoneal exudation remains sterile,

a post-mortem examination of the animals enables one to recognise,

beneath the peritoneal surface, small white points made up of

leucocytes. The sowing of these masses almost invariably gives colonies

of the _Bacillus pyocyaneus_ which form blue pigments. We see from this

account that, even in the peritoneal cavity of vaccinated animals,

matters by no means go on in a uniform fashion, as would appear from

Wassermann’s statements. Some bactericidal action in the peritoneal

fluid there certainly is, but it is quite transient, and is limited to

the period of phagolysis. The majority of the bacilli resist this attack

of the body fluids to continue their struggle with the phagocytes,

which, however, ultimately get the upper hand. In the subcutaneous

tissue the part played by this phagocytic reaction is still more

general. Gheorghiewsky has studied it not only in vaccinated guinea-pigs

but also in a goat which had received several large injections of the

_Bacillus pyocyaneus_. He observed that shortly after the subcutaneous

injection of these bacilli, the fluid which accumulates at the seat of

inoculation renders them motionless and in part agglutinates them. This

fluid is clear and contains a few leucocytes and a number of bacilli

which still retain their usual form. Some time later the leucocytes

begin to come up to the seat of inoculation and to ingest the bacilli.

At the end of 10 to 15 hours all the bacteria have been seized by the

microphages and we no longer find any of them free. A hanging drop of

this exudation, transported to the incubator, soon swarms with bacilli

which have sprung from the organisms ingested by the leucocytes.

The exudation becomes more and more abundant at the seat of inoculation

and ends in the formation of an abscess, from the contents of which

cultures of the _Bacillus pyocyaneus_ may be obtained for a fortnight.

The bacilli, however, finally disappear, this being due to the

destructive action of the phagocytes and not to that of the fluid of the

exudation.

This fundamental part played by phagocytosis in acquired immunity

against the _Bacillus pyocyaneus_ has been confirmed by Gheorghiewsky by

experiments on guinea-pigs vaccinated and then submitted to the action

of opium. As in the analogous experiments of Cantacuzène on the cholera

vibrio, the opium narcosis retards diapedesis and this, for some time,

increases the chances of the bacilli. A tardy diapedesis and

phagocytosis, no doubt, is produced which ends in the ingestion of the

bacilli, but the animal loses its acquired immunity and finally succumbs

in spite of the fact that the dose of _Bacillus pyocyaneus_ was

insufficient to kill a control guinea-pig vaccinated to the same degree,

but not submitted to the action of opium.

[Sidenote: [249]]

The example we have just analysed relates, then, to a micro-organism

which is more resistant than are the vibrios, Obermeyer’s spirilla or

even the typhoid bacillus, to the action of the microcytase which has

escaped from the cells during phagolysis. The _Bacillus pyocyaneus_

undergoes, in the fluids of the vaccinated animal, the action of the

specific fixative and can thus be rendered motionless and become

agglutinated. But this action is insufficient to ensure immunity and

should phagocytosis not take place in time to ingest the bacilli, the

vaccinated animal succumbs. The reaction of the phagocytes is,

therefore, indispensable if the acquired immunity is to be effective. In

this respect the analogy is very great between the resistance of the

vaccinated animal against the various bacteria (vibrios, spirochaetes,

typhoid cocco-bacilli, bacilli of blue pus) that we have so far studied

in this chapter. These bacteria have, however, this in common;—they are

all endowed with a considerable power of motion. Pursuing our

examination of the principal data on acquired immunity against

micro-organisms, we must now choose examples from the group of

non-motile bacilli; amongst these we assign the first place to the

micro-organism of swine erysipelas. This small bacillus has been the

subject of several important researches on acquired immunity, one of

which at a certain period caused quite a sensation in the

bacteriological world. Emmerich[338], in an investigation carried out in

collaboration with di Mattei, made an unexpected announcement. He said

he believed that he was justified in affirming that the acquired

immunity of rabbits against the bacillus of swine erysipelas is due to

the formation, in the fluids of the body, of an antiseptic substance

which very quickly destroys this organism. This substance, secreted by

the cells of the vaccinated animal, was supposed to act after the

fashion of a solution of bichloride of mercury and to kill a large

number of bacilli, introduced subcutaneously, in from 15 to 25 minutes.

This discovery was not confirmed. In a series of experiments that I

carried out[339] with the object of clearing up this question, and made

under conditions as favourable as possible for the demonstration of the

supposed bactericidal secretion, this action was never manifested. Not

only did the virulent bacilli of swine erysipelas, when injected

subcutaneously into well vaccinated rabbits, remain alive in the

subcutaneous exudation for hours and even days, but the attenuated

bacilli of Pasteur’s vaccines likewise remained intact. These bacilli

when introduced into the anterior chamber of the eye survived for even a

longer period. Here, as beneath the skin, the injection of the bacilli

induced an exudation rich in leucocytes, amongst which microphages

predominated. These phagocytes at once began to seize the bacilli which

were destroyed not in the fluid of the exudation but within the

leucocytes. Long after all the bacilli had been ingested, 24 hours and

more after inoculation, the sowing of the exudation frequently gave

growths in appropriate media.

[Sidenote: [250]]

Emmerich[340] sought by new experiments to remove these objections, but

he found that the bacilli of swine erysipelas did not disappear from the

vaccinated animal until some 8 or 10 hours after they had been

introduced. There is, therefore, no longer any question of a rapid

bactericidal action at all comparable to that of corrosive sublimate,

which would destroy the introduced bacilli in less than an hour. The

limit of 8 to 10 hours, accepted by Emmerich, is still too short and is

not in accordance with my experiments; but even this was quite

sufficient for the appearance of a free phagocytosis, a condition that

really occurs. Emmerich has not directed his researches in this

direction, and his theoretical conclusions did not in the least weaken

the value of my arguments drawn from the demonstration of the ingestion

and intracellular destruction of the bacilli by phagocytes.

[Sidenote: [251]]

The researches on immunity against swine erysipelas then languished for

some time, until the discovery of Pfeiffer’s phenomenon gave a fresh

stimulus to the study of this problem. One of Pfeiffer’s pupils,

Voges[341], sought to apply the results obtained in the case of the

cholera vibrio to the acquired immunity against the bacillus of swine

erysipelas. He studied the blood serum of animals vaccinated against

this bacillus and believed himself justified in affirming the existence

of an acquired bactericidal power. Under no condition, however, did he

observe anything comparable to Pfeiffer’s phenomenon, and he was

compelled to admit that the bactericidal action of the serum is very

feeble and only takes effect on young bacilli whose membranes are as yet

very delicate and not very resistant. Mesnil[342] repeated these

researches in my laboratory, but his results were very different from

those obtained by Voges. The blood serum of rabbits, fully vaccinated

against the bacillus of swine erysipelas, proved to be a good culture

medium for this bacillus, and Mesnil affirms, as the result of numerous

well-established observations, that “_in vitro_, the serum of rabbits

immunised against the erysipelas has no bactericidal power or a very

insignificant one.” On the other hand, the same fluid had a very marked

agglutinative power. The bacillus of swine erysipelas, being non-motile,

does not present the abrupt change that is observed in vibrios or in the

typhoid bacillus when submitted to the influence of specific

serums—under which conditions these organisms at once lose their

motility. But the bacilli of swine erysipelas, when introduced into the

specific serum of vaccinated animals, run together into masses which

become more and more voluminous and fall to the bottom of the vessel,

leaving a limpid supernatant fluid. When this bacillus is sown in the

serum of vaccinated animals, it is seen to develop in the form of

chains, composed of a large number of segments, which fall to the bottom

of the tube. These bacilli, however, whether agglutinated or developed

in chains, never show any attenuation in virulence. When the serum which

bathes them is got rid of by washing, they are just as virulent as are

the bacilli developed in the serum of normal unvaccinated rabbits. It is

important to show that this virulence is kept up in spite of the fact

that the bacilli, when placed in contact with the serum of immunised

animals, become permeated with the specific fixative, as shown by the

experiments of Bordet and Gengou[343]. These observers, indeed, have

demonstrated that the bacilli of swine erysipelas, when kept for 24

hours in the specific serum heated to 55° C., acquire the property of

absorbing the cytases contained in the unheated serum of normal animals.

The study of acquired immunity against the bacillus of swine erysipelas

teaches us that this immunity is not due to any extracellular

destruction comparable with Pfeiffer’s phenomenon; and that this

immunity causes the production of a specific fixative and of a specific

agglutinative substance, whose action on the resistance of the animal,

to judge from the complete virulence of the bacilli when agglutinated

and impregnated by fixative, is feeble or _nil_. It is the phagocytic

reaction which is dominant in the immunised animals and which brings

about the intracellular destruction of the bacilli.

The history of the anthrax bacillus, another representative of the group

of non-motile bacilli, is particularly interesting, the more so that for

some time the researches on acquired immunity have been concentrated

almost entirely on the analysis of the facts observed in animals that

have been vaccinated with the two Pasteur vaccines. In this way a large

number of valuable facts have been collected; of these the more

important may be presented to the reader.

[Sidenote: [252]]

In my first work on this subject[344] I called attention to the fact

that in the rabbit vaccinated against anthrax, the bacilli, when

inoculated subcutaneously, soon become the prey of leucocytes which

accumulate at the spot menaced. In the unvaccinated control rabbits,

however, the anthrax bacilli remain in a free state in the fluid of the

subcutaneous exudation, only a few isolated rods being found inside

phagocytes. I have since been able to confirm this fact[345], which must

now be regarded as fully established. In the vaccinated rabbits the

leucocytes exhibit a very marked positive chemiotaxis against the

anthrax bacilli, whilst in normal unvaccinated rabbits the chemiotaxis

of the leucocytes in the anthrax of the subcutaneous tissue is

distinctly negative. When a small quantity of anthrax culture is

inoculated subcutaneously into vaccinated and into unvaccinated rabbits

there may be observed, even within a few hours, a very great difference.

In the former there is found at the seat of inoculation an infiltration

which swarms with leucocytes in the act of devouring bacilli. In the

normal, susceptible rabbit, on the other hand, the exudation produced is

soft, rich in fluid, and very poor in leucocytes. The vessels in the

vicinity are distended with blood, and the fact that the leucocytes do

not come up to the seat of inoculation is in no way due to the absence

of vascular dilatation which might prevent diapedesis. The vessels are

much more dilated than in the vaccinated rabbit, and yet in the latter

the emigration is incomparably greater. This essential difference must

be attributed to the sensitiveness of the leucocytes, which exhibit a

negative chemiotaxis in the normal rabbit but a very marked positive

chemiotaxis in the vaccinated rabbit.

It has been shown repeatedly that the subcutaneous exudation, very rich

in leucocytes which have had time to ingest all the bacilli, when

inoculated into guinea-pigs, ensures the appearance in them of a

generalised and fatal anthrax; this affords evidence that the

phagocytosis is exercised against virulent and therefore living bacilli.

Marchoux[346], in Roux’s laboratory, has carried out numerous

experiments on the vaccination of rabbits and has observed that the

inoculated anthrax bacilli cause an exudation very rich in leucocytes,

and that these cells ingest and destroy the bacilli. The phagocytes

easily rid the refractory animal of the bacilli in the vegetative state,

but the spores are much more resistant. After being devoured by the

leucocytes they may remain inside them for months without germinating.

Marchoux obtained cultures of anthrax from the subcutaneous exudation

taken from vaccinated rabbits 70 days after inoculation.

[Sidenote: [253]]

The fact that the bactericidal action of the blood serum on anthrax

bacilli is specially well marked in the rat, suggested the idea of

trying to obtain, in this rodent, an augmentation of this property as a

result of vaccination. Sawtchenko[347] attempted to do this in an

investigation already cited in Chapter VI, carried out in my laboratory.

He succeeded in thoroughly vaccinating white rats against virulent

anthrax and in showing that the blood serum of these animals rendered

refractory “is bactericidal in the same degree as that of non-immunised

rats.” In the vaccinated rats “the subcutaneous exudation was as free

from bactericidal substances as was the lymph of the control animals.”

Sawtchenko was unable to demonstrate any increase of bactericidal power

except in the peritoneal exudation of rats vaccinated by injection of

cultures into the peritoneal cavity.

In spite, however, of the absence of any increase in the bactericidal

property of the blood serum and of the subcutaneous exudation in

vaccinated rats, the cell reaction obtained in them is very different

from that met with in normal, susceptible rats. In a very short time (3

to 5 hours) after the subcutaneous injection of anthrax bacilli into the

control rats (susceptible), an evident oedema is produced; in the

vaccinated rat there is none. The exudation, not very abundant in the

latter, already contains a number of leucocytes which are actively

phagocytic, whilst in the control animal, examined simultaneously,

“leucocytes are rarely met with, and few of them contain bacilli.”

Later, the difference becomes still more marked. Pronounced oedema

occurs in the control animal, it is poor in leucocytes but rich in

bacilli, which continue to multiply; but “in the immunised rat, we find

not a clear exudation but a thick and purulent fluid, full of

leucocytes.” These cells devour all the bacilli; not a single one

remains free. “Even after 14 hours bacilli ingested by the leucocytes

are present and a culture of anthrax bacilli may be obtained from fluid

taken from the seat of inoculation. Further, guinea-pigs or rats, when

inoculated with a drop of this exudation (which contains no anthrax

spores), succumb to anthrax.”

[Sidenote: [254]]

Even before these researches on the immunity of rats had been carried

out, an attempt had been made to gain some idea of the differences

presented by the vaccinated fluids of animals as compared with those

presented by the fluids of control animals susceptible to anthrax. In

1886 I was able to demonstrate[348] that the anthrax bacillus develops

abundantly in the defibrinated blood of sheep that had acquired immunity

as the result of vaccination by Pasteur’s method. When these bacilli

contain spores and are inoculated into rabbits they rapidly produce a

fatal anthrax; but when no spores are present the injection of bacilli

does not produce a fatal disease, and such infection is well supported

by the rabbits. From this I concluded at that time that the anthrax

bacillus must, in the blood of the vaccinated sheep, undergo a real

attenuation in virulence, an interpretation which, as will be seen in

the next chapter, was found to be erroneous.

Nuttall[349] showed that the defibrinated blood of refractory sheep

acted as a nutrient medium for the anthrax bacillus. Making comparative

investigations, by the plate method, on the bactericidal power of the

blood of vaccinated and normal sheep, he observed that, in both cases,

there was, at first, a certain decrease in the number of bacilli sown,

more marked in the blood of the vaccinated than in that of the control

animals. Nevertheless, 8 hours after the commencement of the experiment

the anthrax bacteria had produced innumerable bacilli in the blood of

the refractory sheep. Nuttall satisfied himself that this feeble

bactericidal power was not to be compared with the very much greater

power of the blood of the rabbit, an animal specially susceptible to

anthrax.

More recently the properties of the serum of sheep which have been

vaccinated against anthrax have been studied very carefully by

Sobernheim[350]. He also was able to show that this serum allows of an

abundant development of the bacillus, and that, outside the animal, it

does not exercise any more appreciable bactericidal power than does the

serum of the normal sheep. The serum of the best vaccinated sheep was

found to be incapable of destroying even very small quantities of

anthrax bacilli. The only change that Sobernheim could make out was with

regard to the thickening of the bacterial membrane. This modification,

however, was not constant and could not be seen in the serum of certain

vaccinated sheep.

[Sidenote: [255]]

The serum of the sheep vaccinated by Sobernheim exhibited no increase of

agglutinative power as regards virulent bacilli. Gengou[351], however,

made it clear that repeated injections of cultures of the first vaccine

of Pasteur into dogs produced a marked augmentation of this

agglutinative power; but it was only produced when the attenuated

bacillus was used. The virulent anthrax bacillus, developed as isolated

rods, was not affected in the least by serum that was highly

agglutinative for the bacillus of the first vaccine. Gengou also made

the converse experiment with the serum of a dog into which he had

previously injected a number of virulent anthrax bacilli. The dog,

naturally refractory to anthrax, resisted the inoculation perfectly, but

its serum did not acquire any agglutinative power against the first

vaccine. He concluded therefrom that “the part played by agglutinins in

the defence of the animal must be regarded as extremely problematical”

(p. 339). On the other hand the phagocytic reaction in the vaccinated

sheep is always very pronounced and constant. Von Behring[352], in one

of his most recent publications, expresses the opinion that this example

of acquired immunity must be placed in the category of phagocytic

immunity.

In the group of bacilli, several examples of which we have studied, the

typhoid bacillus approaches still more closely to the vibrios and

spirilla in its relation to humoral properties. Here may be observed a

kind of attenuated Pfeiffer’s phenomenon and somewhat profound

modifications taking place under the influence of the serum of

vaccinated animals. The _Bacillus pyocyaneus_ is more resistant to the

injurious influence of fluids taken from immunised animals. This

resistance is still more marked in the bacillus of swine erysipelas and

again still greater in the anthrax bacillus. Whilst, however, these

properties of the fluids of the body are found to be very variable and

of unequal power, the phagocytic reaction is constantly manifested and

always very actively. The leucocytes which, in susceptible animals,

exhibit a very marked negative chemiotaxis or only a tardy and

incomplete positive chemiotaxis, have, in the vaccinated animal, this

positive susceptibility developed in a very high degree.

[Sidenote: [256]]

Before quitting the group of bacteria we must cast a glance at the

mechanism of acquired immunity against representatives of the group of

spherical micro-organisms. Amongst the cocci the streptococci have been

especially studied as regards this immunity. For long great difficulties

were encountered in vaccinating animals against these chain cocci, but

Roger[353], Marmorek[354], Denys and Leclef[355] overcame these

obstacles and succeeded in immunising the rabbit, one of the most

susceptible species, to their pathogenic action. More recently the

larger mammals, notably the horse, have been successfully immunised. A

certain number of important facts, the knowledge of which is useful to

complete the survey of the phenomena of acquired immunity, have thus

been collected.

[Sidenote: [257]]

Roger set himself to study the properties of the blood serum of rabbits

vaccinated against the streptococcus, and established the fact that this

fluid had not the slightest appreciable bactericidal action; the

streptococcus grew in it just as well as in the serum of fresh

unvaccinated rabbits. When, however, he injected cultures grown in the

serum of immunised animals into rabbits, these rabbits did not die and

presented only transient and insignificant lesions. From this fact Roger

concluded that there must be an attenuation of the streptococcus by the

immune serum, a view which was shared by several other observers. In

formulating this view, however, he had not taken into account the

possibility that this serum acted not upon the coccus that had developed

in it but upon the organism of the animal into which it was injected.

Bordet[356], indeed, was able to show that the streptococcus which grows

in the serum of immunised animals is in no way weakened in virulence.

When he took a race very virulent for the rabbit (Marmorek’s

streptococcus) and injected a minimal dose of a culture grown in the

serum of immunised animals, the rabbits died just as did the control

animals, because the amount of serum introduced was too small to exert

any influence. So also, when he filtered this culture and got rid of the

serum bathing the streptococci, it was found to be just as virulent as

that grown in the serum of susceptible unvaccinated animals.

In confirmation of the discovery made by Roger with the serum of

vaccinated rabbits, Bordet showed that the blood serum of horses highly

immunised against the streptococcus did not exhibit any bactericidal

action. Moreover, he found that this serum caused the development of

somewhat agglutinated streptococci and that it was capable of throwing

streptococci grown on the ordinary media into clumps. Summing up his

researches on the properties of this serum Bordet concludes that it

“causes no profound change in the streptococcus. The vegetative

character of the coccus is not appreciably diminished, and its

morphology remains the same save for certain variations in the length of

the chains. Even the agglutinative power, recognised in numerous serums

by recent researches, is, in the antistreptococcic serum, developed but

slightly” (p. 196).

More recently von Lingelsheim[357] has studied the properties of the

serum of animals which he had thoroughly vaccinated against the

streptococcus. He observed a certain slowing of the development of the

coccus in this serum as compared with the growth in cultures made in the

serum of normal, susceptible animals. But this retardation was slight

and transient, and exhibited itself especially in serums to which von

Lingelsheim, following Denys, had added leucocytes.

[Sidenote: [258]]

Von Lingelsheim also noted a certain degree of agglutination of the

streptococcus by the serum of vaccinated animals, although this was much

more feeble than in the case of the cholera vibrio or the typhoid

bacillus, when agglutinated by their corresponding serums. Speaking

generally, he regarded the direct action of the body fluids as

insufficient to bring about the rapid destruction of the streptococci in

the vaccinated organism. “Since the action of the bactericidal

substances is limited in time, the streptococci are able to adapt

themselves to these substances and recover their former energy. As the

phenomena of extracellular solution, of such a form as those observed

under the influence of the cholera antibodies, are absent in the case of

the streptococcus and as, on the other hand, a considerable ingestion of

these organisms by the leucocytes is observed ... we must seek in the

activity of these cells a second important element of the defence of the

animal organism” (p. 78).

[Sidenote: [259]]

To Salimbeni[358], who has carried out in my laboratory an investigation

on this subject, we are indebted for the most reliable information on

the phagocytic reaction in acquired immunity against the streptococcus.

He studied specially the phenomena in the subcutaneous tissue of a

horse, hypervaccinated against Marmorek’s streptococcus; this animal

received in all, at several injections, about five litres of living

culture. In spite of this refractory condition, an oedema at the point

of inoculation was soon produced; in this the micro-organisms remained

free and the leucocytes were sparse. But the cellular reaction, at first

insignificant, developed with great rapidity and many leucocytes,

amongst which the macrophages were much the more numerous, were

attracted. The phagocytosis was still delayed for some time, but it

continued to increase and 20 to 24 hours after the inoculation it was

complete. As soon as the phagocytosis was well established the oedema

began to disappear. In the thick exudation, containing a mass of

leucocytes, the macrophages are filled with a very large number of

streptococci packed together. These cocci develop inside the cells,

cause them to burst and again become free. A fresh arrival of

leucocytes, however, takes place, this time mainly microphages. These

microphages seize the free streptococci that have struggled so

victoriously against the macrophages; this second phagocytic phase is

final. The streptococci still remain alive inside the microphages for

some days, but ultimately are killed and digested by the phagocytes. At

a period when, 5 or 6 days after injection, insignificant or isolated

traces of streptococci are to be found in the microphages, the exudation

when sown in nutritive media still gives abundant cultures. The

incidents of this struggle between the streptococcus and the animal

organism demonstrate the important part played by the phagocytes. The

fact that the macrophages perish and allow the cocci to escape, clearly

proves that these cocci have been ingested alive and virulent, and

consequently that the fluid of the exudation was incapable of destroying

or even of attenuating them. The macrophages, also, were powerless to

bring about this result and the intervention of the microphages was

necessary to cause the disappearance of the cocci. It is, however,

always the phagocytes which ensure the final resistance of the animal.

In presence of these very precise results obtained from the work of

Salimbeni, a work which I followed very closely, the previous researches

by Denys and Leclef (_l.c._) made under less favourable conditions on

vaccinated rabbits are deprived of their importance. These observers

wished to get an idea of the difference between the reactions of the

animal organism (_a_) after the injection of streptococci into the

pleural cavity of immunised rabbits, and (_b_) after injection into that

of normal susceptible rabbits. They killed the inoculated animals and

found a very marked diminution of micro-organisms in the pleuritic

exudation of the former. This diminution could not be attributed to a

lysis of the streptococci by the body fluids, because there were never

any signs of such destruction. Nor could the phagocytosis, very feeble

at first, be considered as the cause of the disappearance of a large

number of the streptococci. Denys and Leclef put forward a third

hypothesis, which attributed this disappearance to the rapid resorption

by the lymph stream of the injected fluid containing the organisms.

Going over the record of their experiments it will be seen that in

vaccinated rabbits the quantity of pleuritic exudation was always very

much less than in normal rabbits. In presence of this feature there is

reason to ask whether, in the case of the streptococci, a large number

of these organisms were not fixed, along with the leucocytes, on the

walls of the pleura, as in guinea-pigs that are inoculated

intraperitoneally? Instead of being satisfied with merely examining the

fluid exudation, the surface of the pleura should have been scraped in

order to ascertain whether the phagocytic reaction was localised in this

region.

In any case such incomplete results on the active immunity of rabbits in

no way weaken the positive results obtained in the subcutaneous tissue

of the horse, in which the phagocytic reaction plays a really

preponderant part.

This example of the streptococci completes our series of bacteria in

which we have studied their relations with the properties of the animal

organism that has acquired immunity. We have still to see whether the

acquired immunity against micro-organisms of animal origin is subject to

the same law as that against bacteria.

[Sidenote: [260]]

For some years past a zealous study of the infectious diseases produced

by animal micro-organisms has been carried out. Besides malaria, which

occupies a most important position, attention has been directed to

certain diseases in domestic animals that are set up by endoglobular

haematozoa and by flagellata, and a fairly large number of accurate data

have been collected with regard to Texas fever and its parasite the

_Piroplasma bigeminum_, as well as upon the epizootic diseases due to

_Trypanosomata_ (Tsetse fly disease or Nagana, “Dourine,” etc.).

We are indebted to Smith and Kilborne[359] for the earliest information

concerning the acquired immunity of Bovidae against Texas fever. R.

Koch[360] then added some very precise observations on the immunity of

calves which had been inoculated with parasites attenuated in the body

of the tick (_Boophilus bovis_). Lignières[361], who devoted much

attention to this question in the Argentine Republic, has discovered a

sure method of vaccinating the Bovidae against the “Tristeza,” the local

name for Texas fever. He brought to Alfort specimens of attenuated

haematozoa, and in Nocard’s presence performed successful vaccination

experiments. Lignières is now engaged in devising a practical method of

ensuring immunity under the special conditions found in the home of the

“Tristeza.” Up to the present, however, there are no sufficient data as

to the mechanism of the acquired immunity in this case. We have fuller

information as to the essential phenomena observed in the organism of

the rat vaccinated against _Trypanosoma lewisi_. We owe to Mme. L.

Rabinowitsch and Dr Kempner[362] the first important data as to the

possibility of immunising white or piebald rats against the disease

produced by the flagellated infusorian. They noted that these animals

when inoculated with the blood of grey rats containing _Trypanosomata_

acquire a very transitory disease which, however, confers an immunity

against any subsequent infection. The flagellated organisms disappear

from the blood within a few weeks, after which fresh injections of these

parasites have no pathogenic effect.

[Sidenote: [261]]

Laveran and Mesnil[363] confirmed these observations, and in addition

made careful observations on the mechanism of this acquired immunity.

After making several inoculations with blood containing _Trypanosomata_

into white rats, they made a study of the properties of the blood serum

of these immunised animals. First they established the fact that this

serum exerts no microbicidal action on the _Trypanosomata_, but it

agglutinates them without, however, rendering them motionless:—“The

masses may be resolved into rosettes in which the _Trypanosomata_,

united merely by their posterior extremities, have their flagella free

and motile at the periphery.”

Laveran and Mesnil then studied the phenomena evolved in the refractory

organism. When injected into the peritoneal cavity of immunised rats the

_Trypanosomata_ are not acted upon injuriously by the body fluids. They

are, however, devoured by the leucocytes. Laveran and Mesnil thus

express themselves on this subject: “... we have demonstrated clearly

and repeatedly that the _Trypanosomata_ are ingested alive, perfectly

isolated and very motile, by phagocytes, and we have followed the

details of this process of ingestion which recalls that of the ingestion

of spirilla by the leucocytes of the guinea-pig. We consider, therefore,

that the immunity is phagocytic in character.”

[Sidenote: [262]]

The main facts on acquired immunity established in connection with the

most diverse micro-organisms, facts just described, may already be said

to lead to certain general conclusions. They indicate in the first place

that acquired immunity is accompanied by phenomena more complicated than

those observed in natural immunity. In the two categories of processes

observed in acquired immunity the phagocytic reaction is the only one

that can be said to be constant. We find it in those examples in which

the influence of the fluids of the body is most manifest, as in the

experimental cholera peritonitis of the guinea-pig, as well as in those

cases where the humoral action is most feeble, as in anthrax or in the

_Trypanosoma_ disease of rats. We have, however, still to establish the

relations that exist between phagocytosis and the part played by the

fluids of the immunised animal, in order that we may, as far as

possible, present a general picture of the inner mechanism of acquired

immunity against micro-organisms. To attain this result we must place

the reader in possession of further well-established facts, and we must

postpone its discussion to the following chapter, which will be entirely

devoted to the above-mentioned problem.

CHAPTER IX

THE MECHANISM OF ACQUIRED IMMUNITY AGAINST MICRO-ORGANISMS

Cytases and fixatives.—Only the latter are augmented in the immunised

organism.—Properties of the fixatives.—Difference between them and

the agglutinative substances.—The part played by the latter in

acquired immunity.—Protective property of the fluids of the

immunised organism.—Stimulant action of the body fluids.—The

protective power of serum cannot serve as a measure of acquired

immunity.—Examples of acquired immunity in which the serums

exhibit no protective power.—Phagocytosis in acquired

immunity.—Negative chemiotaxis of leucocytes.—Theory of

attenuation of micro-organisms by the fluids of immunised

animals.—Refutation of this theory.—Phagocytosis acts without

requiring any previous neutralisation of the toxins.—The origin of

the fixative and protective properties of the body fluids.—The

relation between these properties and phagocytosis.—The side-chain

theory of Ehrlich and the theory of phagocytes.

[Sidenote: [263]]

Whilst, in natural immunity against micro-organisms, humoral phenomena

play no prominent part, in acquired immunity these phenomena assume a

much greater importance. The bactericidal power of the fluids of the

body is, in natural immunity, reduced to a mere trace, for it has been

demonstrated that the power of normal serums to destroy bacteria

corresponds to no natural phenomenon of the living organism, but is

dependent upon the presence of cytases which have escaped from the

phagocytes at the time of the formation of the clot _in vitro_ and

separation of the serum. The presence of the fixative, that other

important element in immunity, has been demonstrated in the normal

fluids only in rare cases and in small quantity. The agglutinative

property of these fluids has likewise shown itself to be little

developed and without any importance in natural immunity.

[Sidenote: [264]]

[Sidenote: [265]]

In acquired immunity against micro-organisms, on the other hand, we find

that the bactericidal and agglutinative powers of the fluids of the body

are very greatly increased. With the discovery that the bactericidal

property was so highly developed in the serums of animals that had been

vaccinated against vibrios arose the belief in the acquisition of a new

and purely humoral property. R. Pfeiffer, especially, insisted on the

fundamental difference between the power of the serum of immunised

animals to transform the cholera vibrios into granules and the

corresponding property of normal serums. In the first case Pfeiffer’s

phenomenon exhibited marked specificity; in the second, it was much more

general. A normal serum transforms into granules, indifferently, vibrios

that are very distinct from one another; whilst the serum of an animal

vaccinated against a particular species or race of vibrios gives

Pfeiffer’s phenomenon with this species or race only. Bordet’s[364]

researches have definitely settled this question. This investigator has

shown that Pfeiffer’s phenomenon is produced, with all the usual serums,

by means of the same substances, the cytases (alexine, or complement of

Ehrlich). But in the serum of vaccinated animals there is added to these

cytases the fixative (sensibilising substance of Bordet, immunising body

or amboceptor of Ehrlich) which exhibits specific properties. Having

thus carefully distinguished the two substances that set up the granular

change in vibrios, Bordet shows that in vaccinated animals it is the

fixative which increases in quantity, whilst the cytase remains pretty

much in the same proportions as in the normal animal. He demonstrated,

in fact, that when we take a very small dose of the serum of a

vaccinated animal which by itself is incapable of transforming the

vibrios into granules, about the same quantity of immunised serum or of

normal serum must be added to it in order that Pfeiffer’s phenomenon may

appear. The quantity of cytase, that soluble ferment which is necessary

for the production of the phenomenon, is, therefore, about the same in

the serum of a normal animal as in that of a well-vaccinated animal.

Whilst the cytase does not increase as a result of vaccinal injections,

the fixative, on the other hand, becomes more and more abundant.

Consequently it is this second soluble ferment that impresses its

characters on the blood serum and on some of the other fluids of the

vaccinated animal. It has been pointed out in the preceding chapter that

the fixative is found in the fluid of the oedema of vaccinated animals,

although in less quantity than in their blood serum. It has also been

mentioned that no fixative is found in the aqueous humour of

well-vaccinated animals. It must be admitted that this ferment is not

inseparably bound to the cells which produce it, as is the case with the

cytases. I have already developed, at some length, the thesis that the

cytases remain, in the normal animal, within the phagocytes, and only

escape from them when these cells are destroyed, whether in the living

animal (during phagolysis) or outside the animal (during the preparation

of the serum). Gengou’s experiments with the plasma and the blood serum

of normal animals have completely confirmed the fundamental observations

that the cytases are not found free in the circulating blood. It is

evident that the same law applies also to an animal that has acquired

immunity. For this reason neither Pfeiffer’s phenomenon nor any

analogous process that demands the action of cytases is ever produced in

the anterior chamber of the eye, or in the subcutaneous tissue, or in

oedema either active or passive. Further, it is in virtue of this same

law that Pfeiffer’s phenomenon does not manifest itself even in the

peritoneal cavity or in the blood vessels of vaccinated animals in which

the phagocytes have been protected from phagolysis by previous

injections of various fluids (physiological saline solution, broth,

etc.). It would be very interesting to be able to demonstrate the

absence of cytases in the fluids of immunised animals by experiments of

the same order as those carried out by Gengou with the fluids of normal

animals, but the obstacles to the realisation of this postulate are too

great. We saw when discussing Gengou’s experiments that it is impossible

to obtain _in vitro_ a fluid identical with the plasma of living blood.

The greatest precautions in collecting the blood and in its after

treatment are insufficient to prevent coagulation taking place sooner or

later. It follows that, as there is always a considerable quantity of

free fixative in the plasma of immunised animals, an infinitesimal

quantity of microcytase, set free from the leucocytes, is sufficient for

the production of Pfeiffer’s or any other analogous phenomenon. There

must be a great improvement in the methods of preparation of plasmas

outside the body before it will be possible to undertake successful

researches on the above problem. For the present we must rest satisfied

with other proofs, already numerous and very demonstrative, of the

absence of free cytases in the normal plasmas of vaccinated animals.

[Sidenote: [266]]

The cytases being found in about the same quantity and presenting the

same properties in all animals that enjoy immunity whether natural or

acquired, it must be the fixative which specially distinguishes these

two categories of immunity. Now, the fixative is found in the serum of

perhaps all cases of acquired immunity. Bordet and Gengou have studied

it by the method already mentioned (Chap. VII.). A certain quantity of

micro-organisms of various species is introduced into the serum. If the

cytases, present in the serum when the experiment was commenced,

ultimately disappear from it, it indicates that this ferment has been

absorbed by the bacteria, thanks to the fixative, which consequently

should be found in the serum under observation. The presence or absence

of the cytases can be demonstrated by the production or absence of

Pfeiffer’s phenomenon with vibrios.

The application of this method enabled Bordet and Gengou[365] to satisfy

themselves that the serum of animals immunised against several species

of bacteria (plague bacillus, typhoid bacillus, bacillus of swine

erysipelas, first anthrax vaccine, and _Proteus vulgaris_), really

contains an appreciable quantity of fixative. It may, then, be accepted

that the production of this substance is fairly constant in acquired

immunity against bacteria, and that it constitutes one of the most

important factors in such immunity.

The question has been raised: What is the nature of the substance to

which the name of fixative is given? Pfeiffer and Proskauer[366] have

attempted to solve this question by making use of a serum which acts

against the cholera vibrio and which they obtained by vaccinating

animals with this vibrio. They carried out a long series of experiments

which led them to the conclusion that this substance, which they term

“cholera antibody,” cannot be identified with any of the albuminoid

substances of the serum. Further, the fixative is not represented by any

of the salts or extractive substances of the serum, because these

substances dialyse easily, whereas the cholera antibody does not pass

through the dialysing membrane. The fixative is wholly precipitated by

alcohol, and is regarded by Pfeiffer and Proskauer as belonging to the

category of soluble ferments, an opinion which is certainly shared by

many other investigators.

[Sidenote: [267]]

[Sidenote: [268]]

What is it that communicates to this ferment its remarkably specific

character? Without being able to give a precise answer to this

question, the authors just cited point out the analogy that exists

between the cholera antibody and the soluble ferments of yeasts which

have been studied by Emil Fischer. Some of these act only upon certain

special sugars in a manner equally specific. From a logical point of

view it might be permissible to attribute the specificity of fixatives

to something borrowed from the species of micro-organism that has

played a part in their production. It has long been recognised that in

old cultures of the cholera vibrio these micro-organisms are

transformed into spherical granules, the arthrospores of Hueppe, which

closely resemble the granules produced in Pfeiffer’s phenomenon. There

are, then, undoubtedly, vibrionic products which act much as do the

microcytases, and it would be very interesting if we could find them

in the bactericidal ferments of the animal body. An attempt of this

kind was undertaken by Emmerich and Löw[367], who attribute the

acquired immunity to a particular substance which they term

“Nuclease-Immunproteïdin.” According to their hypothesis the microbial

products which are produced in the animal during the period of

vaccination—the nucleases—combine with proteid substances of the blood

and organs to furnish the substance to which these authors have given

such an elaborate name. In their most recent publication Emmerich and

Löw even describe a method of producing this substance outside the

animal body, by the action of ox blood, or better still pounded

spleen, on the nuclease produced by the bacteria found in old

cultures. To it they attribute the property of dissolving the various

bacteria, of conferring immunity against and even of curing several

infective diseases. But these authors do not say whether this

remarkable substance is identical with, or analogous to, the

antimicrobial ferments composed, as we have seen, of microcytase and

fixative. It must be concluded that they look upon it as being similar

to the alexine of Buchner, which is nothing more than a mixture of the

two substances just named. Unfortunately the whole account given by

Emmerich and Löw will do anything but gain over the reader, and in

their publications no proof of their assertions can be found. Several

of the facts advanced by them do not fall in with well-established

data. Thus they speak of the complete lysis of the bacilli of swine

erysipelas by their soluble “Erysipelase-Immunproteïdin” in vaccinated

animals, a process that has never been demonstrated by them and which

in no way accords with conscientious and carefully carried out

observations. On the other hand, they cite facts which contradict one

another. The “Pyocyanase-Immunproteïdin” is a substance which

possesses an extraordinary bactericidal power, not only against the

_Bacillus pyocyaneus_ but also against several other bacteria, _e.g._

the bacilli of anthrax, diphtheria, typhoid, and plague. This

substance rapidly breaks up these bacteria, and cures diphtheria and

experimental anthrax. But it is, at the same time, so affected by the

invasion of the most common bacteria, such as _Bacillus subtilis_,

that it is necessary to add antiseptics in order to preserve it. To

these contradictions, inaccuracies, and uncertainties must be added

further the advice, given by Emmerich and Löw to bacteriologists, not

to attempt to reproduce their experiments, because they may easily

fail, and I think that, in spite of the seductiveness of the attempt

to attribute to bacterial products a share in the elaboration of

antimicrobial substances, we must conclude not to follow these authors

further. It is better to confess our ignorance of the chemical

composition of these substances in general and of the fixatives in

particular.

[Sidenote: [269]]

As the fixatives resist temperatures much higher than those which

destroy the cytases, in this respect resembling the agglutinative

substances so frequently found in the fluids of vaccinated animals,

there has long been a tendency to identify them with these latter. It is

indisputable that between the fixatives and the agglutinative substances

the analogies are fairly numerous. Both are produced in quantity during

the process of immunisation, and are found not only in the blood serum

but also in the fluids of the living animal, especially in the fluids of

exudations and transudations. Both dialyse through parchment more

readily than do the cytases. Buchner[368] has demonstrated that his

alexines (bactericidal substances of normal serum) will dialyse only

where the lower fluid is pure water; dialysis is _nil_ when the

distilled water is replaced by physiological saline solution. The

fixatives and agglutinins, as demonstrated by Gengou[369] for the

latter, pass almost completely through the dialyser in the case of pure

water, and one-half still passes when the lower fluid approaches as

nearly as possible to normal serum.

In spite of these analogies, however, the agglutinative property must be

sharply distinguished from the fixative power of serums. In this fluid,

derived from normal animals, the agglutinative property is often very

marked when the power of fixing the cytases is totally, or in great part

absent. Bordet and Gengou[370] have demonstrated also that feebly

agglutinative serums of persons convalescent from typhoid fever may

exhibit a great capacity for fixing the cytases. Other facts, to be

mentioned later, confirm the real difference between the fixative and

the agglutinative properties.

[Sidenote: [270]]

The agglutination of bacteria was noted during the course of a series of

researches on the acquired properties of the blood serum of vaccinated

animals. Charrin and Roger[371], seeking to obtain a clear idea of the

difference between the serum of normal animals and that of animals

vaccinated against the _Bacillus pyocyaneus_, observed that this

bacillus developed in the normal fashion in the former, but in the

latter gave rise to special forms of growth. Instead of growing in the

form of rods, it elongates into segmented filaments which become

entangled and fall to the bottom of the tubes, leaving a supernatant

limpid serum. I was able not only to confirm the accuracy of this

observation for the _Bacillus pyocyaneus_, but to extend it to

Gamaleia’s vibrio and to the pneumococcus[372]. In all these instances

we have a modification of the bacteria developed in specific serums

coming from vaccinated animals. Later, Bordet[373], during his

researches on the bacteriolysis of vibrios _in vitro_, observed that

these vibrios, when introduced into the blood serum of vaccinated

animals, lose their movements and soon unite into more or less

voluminous masses. This observation was confirmed by Gruber and

Durham[374], who were the first to apply it in the specific diagnosis of

bacteria. They showed that the agglutinating power of vaccinated

animals, although not rigorously specific, might, nevertheless, be

utilised for the differentiation of certain bacteria, especially the

cholera vibrio and the typhoid bacillus. But, independently of this

result, Gruber[375] essayed to formulate a theory of acquired immunity

based on the agglutinative property of the serum. He accepted, in

connection with the phenomenon of the destruction of the bacteria,

Bordet’s hypothesis of the concurrent action of two substances, of which

one, the bactericidal substance proper, is nothing but the alexine of

Buchner, the second being that which agglutinates the bacteria. This

agglutination, according to Gruber, results from the swelling of the

bacterial membrane which becomes viscous and so leads to the cohesion of

the bacteria and the formation of clumps. Thus transformed and rendered

motionless, the bacteria succumb more readily to the destructive action

of the alexine. It is supposed that the phagocytes do not intervene at

all in these cases of acquired immunity, except in a purely secondary

fashion when they ingest the bacteria already greatly weakened by the

united action of the agglutinin and the alexine. The principal _rôle_ in

this theory of immunity is thus given to the agglutinative substance,

which is regarded as being a microbial product, modified by the

macrophages and thrown into the blood.

[Sidenote: [271]]

The discovery of this agglutination of bacteria has acquired great

importance, especially in connection with its application to the

diagnosis of typhoid fever. Widal[376] succeeded in showing that typhoid

bacilli agglutinate readily under the influence of blood serum and other

fluids (milk, transudations, tears, etc.) derived from patients

suffering from typhoid fever. As this phenomenon could be utilised for

the early recognition of the disease, it began to be studied with great

care and many interesting data concerning it have been collected. The

general outcome of these researches accords with the conclusions drawn

by Widal, and the serum-diagnosis of typhoid fever has taken an

important place among the methods used for the recognition of this

disease. This aspect of the question, however, does not interest us from

the point of view of the problem of immunity which we now have under

consideration, and we cannot here enter upon the study of the

serum-diagnosis of typhoid fever and certain other diseases (cholera,

tuberculosis, pneumonia). Moreover, we must refrain from any analysis of

the hypotheses advanced to explain the mechanism of agglutination. A

lively discussion has been carried on between the partisans of the

chemical theory—according to whom the agglutinin acts directly on the

agglutinable substance of the bacteria—and the advocates of the physical

theory, led by Bordet[377], who attribute the agglutination to

modifications in the molecular attractions which unite the agglutinable

elements, be it between each other or with the surrounding fluid. At one

time it was thought that Roger’s[378] observation that the cell

membranes of _Oïdium albicans_, when cultivated in the specific serum of

immunised animals, increased in volume and became greatly swollen,

settled the question in favour of Gruber’s theory. But the objection

formulated by Kraus and Seng[379], on the one hand, and by Bordet, on

the other, dealt a severe blow to this view. As the serum employed by

Roger was not deprived of its cytases (alexine), the viscosity of the

membrane of the fungus could not be attributed to the agglutinin. When

Bordet[380] demonstrated that the red blood corpuscles, under the

influence of the serums, undergo an agglutination as marked as that seen

in bacteria, it enabled us to study this phenomenon in the large red

corpuscles of birds, in which no one has ever been able to demonstrate

any viscosity of the corpuscular stroma. In a mixture of red corpuscles

of bird and mammal, submitted to the action of a serum which

agglutinates the former only, the red corpuscles of the mammal never

unite with those of the bird, although this should undoubtedly take

place if the membrane of the agglutinated corpuscles had really become

viscous. All the facts collected up to the present are, therefore, in

favour of Bordet’s physical theory in which an analogy between the

phenomena of agglutination and of coagulation is traced.

[Sidenote: [272]]

The point that interests us more particularly in regard to agglutination

is the relation of this phenomenon to immunity. We have already given

(Chapter VII) the arguments which render it impossible for us to

attribute to the agglutinative property of the fluids of the body any

_rôle_, however unimportant, in natural immunity against

micro-organisms. We must now study the importance of this property in

the condition of acquired immunity, in which the agglutination of

micro-organisms by the fluids of the body is much more frequent and

active than in natural immunity.

[Sidenote: [273]]

The first question to be settled is the following: Is the agglutinative

property really constantly present in the fluids of vaccinated animals?

The blood serum of animals that have acquired immunity is unquestionably

usually agglutinative as regards the corresponding micro-organism. This

agglutination may be more or less pronounced, but it certainly exists in

the great majority of cases. Nevertheless, examples can be cited in

which, in spite of the refractory condition acquired as the result of

immunisation, the serum exhibits not a trace of agglutinative power.

Having demonstrated that several bacteria (_Bacillus pyocyaneus_,

_Diplococcus pneumoniae_, _Vibrio metchnikovi_) develop in the serum of

vaccinated animals in the form of elongated filaments more or less

interlaced, I was quite prepared to allow that this fact might be of

general import. But the study of a cocco-bacillus which produces the

pneumo-enteritis of swine and which was isolated by Chantemesse during

an epizootic at Gentilly, led me to believe that this was not the case.

As this bacillus is characterised by great motility, I concluded[381]

that it was identical with that of the hog cholera of American writers.

Theobald Smith[382], to whom I sent a specimen and who is a competent

authority on this question, refers it, however, to the species which

produces swine plague. Knowing that the question of these two bacteria

is not finally settled, it is impossible to come to an absolute decision

in the matter. Fortunately, from the point of view of immunity, this is

of no great importance. The point upon which I must lay stress is that

the serum of rabbits vaccinated against the Gentilly bacillus, when sown

with this cocco-bacillus, gave very abundant and uniformly turbid

growths. In my researches, undertaken at a period when the rapid

agglutination of micro-organisms added directly to the specific serum

had not yet been recognised, I noted merely that the cocco-bacilli which

grew in the blood serum of vaccinated rabbits presented their normal

form and gave rise to a general turbidity of the fluid. Since then,

however, it has often been observed that the mode of development of a

micro-organism in a serum gives an even more delicate indication than

does the agglutination properly so called, produced by the serum to

which has been added an organism cultivated on its usual medium. Thus

Pfaundler[383] saw that _Bacillus coli_ and _Proteus vulgaris_, which

were not agglutinated by certain serums, developed in them in an unusual

fashion and produced very long and interlacing filaments. When a serum

is incapable of revealing its properties by agglutinative reaction

properly so called, it is sown with the corresponding micro-organism and

the development is then compared with that observed in a normal serum.

Frequently a very marked difference is noted, the same organism growing

into filaments in the specific serum and forming rods only in the normal

serum. The first mode of development is sometimes designated

“Pfaundler’s reaction.”

In the serum of rabbits vaccinated against the Gentilly cocco-bacillus,

no filaments corresponding to those met with in the agglutinative

reaction are formed, but bacilli are produced. In spite of this the

animals that furnish the serum show a distinct resistance to infection.

More recently, Karlinski[384] has studied the properties of the serums

of animals treated with the cocco-bacilli of hog cholera and swine

plague. He was able to demonstrate that blood serum from oxen that had

received repeated injections of cultures or toxin of hog cholera, was

not only incapable of killing the cocco-bacilli of the two swine

diseases but it even “gave rise to no agglutination” of the two bacilli

and did not arrest the motions of those of hog cholera. On the other

hand, serums have been obtained from other species of animals (dog, pig)

which brought about the typical agglutination of the cocco-bacillus of

hog cholera[385].

[Sidenote: [274]]

In the preceding chapter, Gengou’s experiment on the serum of a dog that

had been treated with a virulent culture of anthrax has already been

cited. This serum did not agglutinate the bacillus, even of the first

vaccine of Pasteur. Nevertheless, a second dog treated with an

attenuated culture of this bacillus furnished an agglutinative serum.

The immunisation of the first dog was carried very much further than

that of the second, but the agglutinative properties were in inverse

order. Sawtchenko, in his study of immunity against anthrax,

demonstrated that the subcutaneous exudation from vaccinated rats does

not agglutinate the bacillus which usually exhibits such a great

tendency to collect into clumps.

Agglutination has been studied particularly carefully in typhoid fever.

We know that after an attack of this disease, an acquired refractory

condition is produced which lasts for a considerable period. In most

cases the agglutinative power of the blood diminishes very rapidly, and

disappears a few weeks after the commencement of convalescence. It is

only in rare cases that it persists for years[386]. On the other hand,

during the period of apyrexia which precedes the relapse in typhoid

fever and during the period of relapse, the agglutinative power may

manifest itself in a very marked degree. In an observation made on a

case reported by Widal and Sicard[387], the agglutinative power was

raised, two days before the relapse, to a ratio (1 : 150) it had never

attained during the first attack. “The appearance of the relapse, two

days after this observation”—these authors add—“renders it evident that

the agglutinating reaction is independent of the state of immunisation.”

Analogous cases have been pointed out repeatedly by several observers.

[Sidenote: [275]]

The examples cited show, on the one hand, that the serum of individuals

endowed with acquired immunity may be without any agglutinative

property, but, on the other, that this power may be highly developed in

the serum of susceptible individuals. The argument based on these data

may be corroborated by several other series of facts. Thus,

Salimbeni[388] has pointed out that the cholera vibrio is not

agglutinated in the fluids of immunised animals. The subcutaneous

exudation of a horse treated with a large quantity of these vibrios does

not agglutinate Koch’s vibrio except outside the body. When this

exudation is drawn off shortly after the injection of the vibrios, the

organisms render the fluid uniformly turbid. But a short exposure to the

air is sufficient to bring about the agglutination of the vibrios in the

same exudation. Guided by this observation, Salimbeni carried out

comparative experiments on the action of the serum of vaccinated animals

outside the body, in tubes deprived of oxygen and in others exposed to

the air. In the former agglutination did not take place or was very

incomplete, in the latter it soon came on. This fact accords perfectly

with the observation of Pfeiffer’s phenomenon in the peritoneal cavity

of guinea-pigs from which we withdraw a fluid containing granules that

have resulted from perfectly isolated vibrios. In other micro-organisms

a difference has been noted in this respect. Thus Gheorghiewsky has seen

the agglutination of the _Bacillus pyocyaneus_ produced under the

influence of the serum of vaccinated animals, even in tubes deprived of

oxygen. Durham has made a similar observation in the case of the typhoid

bacillus. When, however, Trumpp[389] wished to satisfy himself as to the

agglutination of the same organism in the body of well-vaccinated

guinea-pigs, he obtained only imperfect results. He concluded from his

experiments “that the formation of typhoid clumps may precede the

breaking down of the bacteria in the animal body itself, but only under

certain conditions—when the degree of immunity of the animal is

sufficiently high and when the bacilli introduced are not too numerous”

(p. 130). In the case of the typhoid bacillus, a certain degree of

agglutination is produced inside the animal body, but it is markedly

increased in the fluids that have been withdrawn and exposed to the

action of the air.

[Sidenote: [276]]

It has been demonstrated, repeatedly, that the agglutination of

micro-organisms by their specific serums does not prevent their growth

and multiplication. These agglutinated organisms lose none of their

virulence. Issaeff[390], working in my laboratory, carried out an

investigation on this point in the case of the pneumococcus. He

vaccinated rabbits against this organism and satisfied himself that the

organism still grows well in the blood serum of such rabbits; but,

instead of presenting the typical form of lanceolate diplococci, the

pneumococcus, under these conditions, forms very long chains of true

streptococci. Having filtered the cultures in order to get rid of the

serum, he injected them into rabbits and mice and demonstrated that the

pneumococci had retained to the full their initial virulence.

Sanarelli[391] carried out corresponding experiments with Gamaleia’s

vibrio, which, as we know, also forms chains in the serum of vaccinated

animals. When filtered on a paper filter and washed with physiological

saline solution, the vibrios were found to be just as virulent as were

the control vibrios grown in the serum of susceptible animals. More

recently, Mesnil[392] demonstrated the same point in connection with the

bacillus of swine erysipelas. He experimented on cultures that were

agglutinated after their formation and also on others agglutinated as

they were growing. The fluid of the culture was decanted and replaced by

fresh broth until the elimination of the serum was complete. Mice,

inoculated with the washed clumps, died in the normal period, thus

affording proof that “agglutination in no way alters the vitality and

virulence of the bacillus of swine erysipelas” (p. 492).

We can readily understand, after the demonstration of these various

facts, that it is impossible to maintain Max Gruber’s theory that the

agglutinative power constitutes the fundamental basis of acquired

immunity. Hence this writer, after publishing several preliminary notes

in 1896, has not yet decided to give to his hypothesis a more extended

development. Nor has any one else attempted to defend it.

[Sidenote: [277]]

It is probable that in certain special cases the immobilisation of very

motile bacteria and their agglutination into clumps may facilitate the

reaction of the animal organism, especially the rapidity of

phagocytosis. Thus, Besredka[393] observed that guinea-pigs when

inoculated with typhoid bacilli that had previously been mixed with the

blood serum of normal animals survived. The most active amongst these

serums was ox serum heated to 60° C. Guinea-pigs furnished a serum which

was much less active. The resistance of guinea-pigs, inoculated into the

peritoneal cavity, was in direct ratio to the agglutinated condition of

the bacilli. Besredka lays stress on the facility with which the

bacilli, when agglomerated into large clumps, were ingested by the

phagocytes, and suggests that there is a certain stimulating action of

the serums on the leucocytes. When he injected into guinea-pigs a

mixture of typhoid bacilli and guinea-pig’s serum, made immediately

before injection, his animals died from infection. But when he left the

bacilli for some time in contact with the guinea-pig’s serum outside the

body, and did not inject the mixture until after agglutination was

complete, the inoculated animals usually survived. This experiment

indicates the part played by agglutination in the resistance offered by

the animal, and at the same time proves that in the body of the

guinea-pig the agglomeration of the micro-organisms into clumps does not

take place to the same degree as in the serum prepared in, and left in

contact with, the air.

In any case, the data collected by Besredka cannot be put forward as an

argument in favour of the essential part played by agglutination in

acquired immunity, nor can they weaken the facts indicated as to the

absence of agglutinative power in examples of acquired immunity and as

to the virulence of the agglutinated micro-organisms. The part played by

agglutination in this immunity is merely accidental and subordinate.

[Sidenote: [278]]

Special researches have been carried out with the object of defining,

exactly, the origin of agglutinins in the body of an animal that has

acquired immunity. Observers are unanimous in recognising that, of all

parts of the organism, the blood is richest in agglutinin. This

substance is found in the blood serum as well as in the plasma. From

this (corroborated by the agglutinative property of other fluids, such

as the pericardial fluid, oedemas very poor in formed elements, etc.) it

follows that the agglutinin circulates in the blood and lymph of the

living animal. Several observers, amongst whom I may cite Achard and

Bensaude[394], Arloing[395], and Widal and Sicard[396], put to

themselves the question whether, before passing into the blood, the

agglutinin is not formed in the exudation developed at the seat of

inoculation of the micro-organisms. Their conclusions were invariably

negative; they were never able to find more agglutinins in these

exudations than in the blood. Pfeiffer and Marx[397] had occasionally

observed that their animals, inoculated with the cholera vibrio, early

exhibited an agglutinative power in the spleen; but this result was not

met with sufficiently constantly to enable them to draw a positive

conclusion. A little later, van Emden[398] studied in detail the

distribution of the agglutinative property in the body of an animal

inoculated with _Bacillus aërogenes_. His researches led him to the

conclusion that the spleen and the lymphoid organs must be regarded as

the source of the agglutinins. Shortly after the inoculation of the

bacilli, an extract of the spleen was more agglutinative than the blood

or any of the other organs. In rabbits from which the spleen had been

removed, the same _rôle_ was filled by the bone marrow and probably also

by the lymphatic nodules. But this preponderance of the haematopoietic

organs did not continue long, the blood soon becoming the most important

seat of the agglutinative power.

The proof that this question of the origin of the agglutinins is a very

delicate and difficult one is afforded by an investigation very

carefully carried out by Gengou[399] on the agglutination of the

attenuated anthrax bacillus (Pasteur’s first vaccine) by the fluids and

organs of normal and prepared guinea-pigs. This observer was never able

to obtain any confirmation of the results obtained by van Emden with

another micro-organism. In Gengou’s guinea-pigs it was always the blood

fluid which showed itself most agglutinative, the organs exhibiting

merely a feeble and inconstant agglutinative power. As the extracts of

leucocytes were always found to be markedly less active than the blood

and the fluids of the exudations, Gengou was obliged to come to the

conclusion that the agglutinins cannot be regarded as products of the

cells of the animal body; this he sums up by saying that “in the

increase of the agglutinative power of its blood the organism of the

animal plays only a relatively passive part” (p. 337).

[Sidenote: [279]]

I think that, in spite of the facts established by Gengou, his

conclusion can scarcely be regarded as final. The agglutinative

property, developing in the animal body, must be attributed to some

cellular influence, because we know that the prolonged sojourn of

micro-organisms in the animal fluids is incapable of conferring on them

this power. As Gengou’s experiments did not permit him to attribute the

formation of agglutinin to any formed element, it must be concluded

that, although perfectly exact, they were insufficient to solve the

problem. Gengou killed his animals at a stage when their blood was

already pretty strongly agglutinative. At this stage the organs only

possessed it to a much more feeble degree. Perhaps, if he had examined

his animals at an earlier stage, when the blood possessed a much less

marked agglutinative power, he might have obtained a more powerful

agglutination with an extract of the organs. In my researches on the

resorption of cells, I observed, on several occasions, that the

abdominal fluid of guinea-pigs which had received an injection of

goose’s blood became agglutinative before the blood serum. Later,

however, the blood exhibited a greater agglutinative power than did the

peritoneal fluid. If to this fact we add the results of van Emden’s

experiments, we shall be tempted to assign to the cells found in the

peritoneal exudation and in the lymphoid organs a share in the

production of the agglutinin. This question of the origin of the

agglutinative power is, however, a very difficult one, and it is

impossible, in the imperfect state of our knowledge, to express oneself

in a more positive fashion. Fortunately, according to the whole of our

data on this phenomenon, the part played by agglutination in immunity

can only be very inconsiderable, and we may be allowed to consider our

general problem without concerning ourselves over much about the origin

of the agglutinative property.

Among the definite results obtained from the study of the agglutinins,

it may be specially pointed out that these substances can in no way be

identified with the fixatives. These latter were, for long, spoken of as

_preventive substances_. They are so termed in the early papers of Jules

Bordet treating upon this question. The explanation of this designation

is that, for a series of years, the presence of the fixatives was

revealed chiefly by the preventive or protective property of the media

which contained them.

[Sidenote: [280]]

To gain a clear conception of this protective property, which occupies

so important a place in the study of acquired immunity, we must go back

to an epoch in our science when it was sought to prove that the fluids

of the body played a part in the production of immunity. Shortly after

the earliest researches on the bactericidal power of the blood had been

made, the idea of applying the results obtained in this direction to the

production of immunity in animals by means of injections of blood

occurred. The first step in this direction was taken by Richet and

Héricourt[400], who succeeded in vaccinating rabbits against a variety

of staphylococcus by means of defibrinated dog’s blood. The dog is

naturally refractory against this organism, and the blood of a normal

dog exercised a certain vaccinal or protective influence on rabbits

inoculated with the staphylococcus. But this action was much more marked

when Richet and Héricourt employed the defibrinated blood of dogs which

had previously received inoculations of the staphylococcus. Shortly

after this observation, von Behring[401] made his discovery of

antitoxins in the blood serum of animals immunised against tetanus and

diphtheria toxins. In collaboration with Kitasato he demonstrated that

the serum of these animals, when injected into normal animals, protected

them against intoxication by the poisons of diphtheria and tetanus. This

great discovery, which has been confirmed on all sides and extended to

other poisons, gave rise to the view that a serum exerting any

protective power depends solely on its property of impairing the action

of the toxins. A more careful study of the phenomena which appear under

the influence of the serums has, however, demonstrated the inaccuracy of

this view. I was able to furnish the proof[402] that the blood serum of

rabbits vaccinated against the micro-organism of the Gentilly

pneumo-enteritis prevented normal rabbits from contracting a fatal

infection. Nevertheless, the serum exerted no influence on the toxin of

this micro-organism; the rabbits that received the minimal lethal dose

of this toxin, mixed with serum from vaccinated rabbits, died, as did

the control animals, from rapid poisoning. It was evident then that this

serum, which prevented infection without in any way hindering

intoxication, could not be classed in the category of antitoxic serums.

We find ourselves, therefore, in the presence of a new property of the

fluids of the body to which we have given the name of _protective_ or

_anti-infective power_. We are driven to this conclusion the more as the

serum in question was neither bactericidal nor agglutinative.

[Sidenote: [281]]

This discovery was soon confirmed by R. Pfeiffer[403] for the cholera

vibrio. Animals vaccinated against this organism furnished Pfeiffer with

a serum which, whilst not at all antitoxic, was distinctly protective

when injected into normal guinea-pigs. It protected these animals from a

fatal infection by the vibrio and, when injected into the peritoneal

cavity, it set up the granular transformation of the cholera

vibrios,—Pfeiffer’s phenomenon. Pfeiffer, for this reason, gave to the

protective antivibrio serum the name of bactericidal serum. As the

granular transformation was produced, under the influence of this serum,

with cholera vibrios only and never with other species of vibrio,

Pfeiffer gave to the active substance in the serum the name of _specific

cholera antibody_. This substance, according to his theory, was formed

in the animal body at the expense of an inactive antibody which became

transformed into an active substance under the influence of the

peritoneal endothelium.

The possibility of thus vaccinating susceptible animals by means of the

serums of immunised animals, quite apart from any antitoxic power, was

easily confirmed and extended to several other infective diseases.

Pfeiffer and Kolle[404], Funck[405], Chantemesse and Widal[406]

demonstrated it in connection with the experimental disease produced in

animals by the typhoid bacillus; Loeffler and Abel[407] for the

_Bacillus coli_, etc. The protective or anti-infective power of the

serum and other fluids of immunised animals was soon recognised as a

general property.

[Sidenote: [282]]

Pfeiffer and his collaborators, as well as many other investigators,

laid special stress on the bactericidal character of these protective

fluids. It was seen that the serums of immunised animals were often

almost or completely incapable of killing the corresponding

micro-organisms, but they were still regarded as bactericidal, because,

when injected into the peritoneal cavity of normal animals, they set up

the transformation of vibrios into granules, or, in the case of other

bacteria, determined certain phenomena of extracellular destruction.

Whilst carrying on researches in this direction, Fränkel and

Sobernheim[408] discovered a fact of great importance. They found that

the protective substance of the serum of animals vaccinated against the

vibrios resisted heating to 70° C. When submitted to the influence of

this temperature, the serum lost its bactericidal power completely, but

remained quite as protective as the unheated serum, when injected into

susceptible animals. This experiment, which has since been confirmed

repeatedly, furnished us with a means of separating the bactericidal

power from the protective power in cases where both were present in the

same serum. Later, in the hands of Bordet, it proved to be of great

service in connection with his researches on the concurrence of two

substances in acquired immunity.

The possibility of obtaining Pfeiffer’s phenomenon outside the body by

“reactivating” the protective serum with peritoneal fluid or blood serum

of normal unvaccinated animals has still further facilitated the study

of the action of the two substances in acquired immunity. It was with

the help of this method that Bordet was able to furnish so much valuable

information on the subject of anticholera serums and, later, on that of

haemolytic serums. The discovery by Ehrlich and Morgenroth[409] of the

fixation by the sensitive elements of the heat-resisting (thermostabile)

substance (that which resists a temperature of 65°–70° C.) constitutes a

new and important acquisition to the study of acquired immunity. The

discovery has been applied by Bordet to micro-organisms, and since then

it has been found possible to study much more precisely the mode of

action of specific protective serums.

[Sidenote: [283]]

Even before this last scientific advance had been made it was possible

to determine the relations between the protective power and the

agglutinative power of the fluids of animals that had acquired immunity.

Both resist about the same temperatures; both are found in the blood

plasma and pass into the fluids of exudations and transudations. But it

may be affirmed with certainty, as already stated, that the two

properties are quite distinct. Pfeiffer has laid great stress on the

fact that highly protective serums often exhibit only a feeble

agglutinative power and _vice versa_. During an investigation[410] into

an epidemic of typhoid fever, he had occasion to study the serum of

patients convalescent from this disease. The exact dosage of the two

properties demonstrated that a slightly marked agglutinative property

might be associated with a very powerful protective property.

Gheorghiewsky[411] made similar observations on animals vaccinated

against the _Bacillus pyocyaneus_. The serum of a goat, although more

agglutinative, invariably proved to be less protective than that of a

rabbit. A similar result was obtained with the serum of immunised

guinea-pigs. “This shows distinctly”—concludes Gheorghiewsky—“that the

property possessed by serums of agglutinating the _Bacillus pyocyaneus_

does not march parallel with the protective property” (p. 304).

Analogous examples are sufficiently numerous to justify us in accepting

the distinctiveness of the two properties of specific serums.

The protective or anti-infective substance is, therefore, not the same

as the agglutinin. But are we justified in regarding it as identical

with the fixative substance, or fixative (sensibilising substance,

immunising or intermediary substance, or amboceptor)? From the fact that

the fixative was at first rightly designated by Bordet as protective

substance we should conclude in the affirmative. The question is an

important one and merits close examination. The discovery of an exact

method of determining the presence of fixatives has rendered it possible

to ascertain whether these substances are always found in the protective

fluids and also whether the presence of fixatives necessarily implies

the protective power of the serums.

The first of these questions has been answered in the affirmative. All

the protective serums studied from this point of view, by Bordet and

Gengou, were found to be endowed with very distinct fixative properties.

They also found the specific fixative in the serum of guinea-pigs

immunised with the attenuated bacilli of the first vaccine of Pasteur.

Now this serum is powerless to prevent the production of fatal infection

in mice into which is simultaneously injected the bacillus of the first

vaccine. Consequently a fixative fluid is not necessarily protective.

This is in accordance with the fact that the micro-organisms that have

absorbed the fixative may, nevertheless, retain their virulence. We have

already cited the experiment of Mesnil that the bacilli of swine

erysipelas, mixed with the specific serum and then deprived of this

fluid, produce a fatal infection in mice. We have also drawn attention

to the fact, demonstrated by Sawtchenko, that anthrax bacilli, obtained

from the exudation of immunised rats, give rise to a fatal anthrax in

normal guinea-pigs and rats. The experiments of Bordet and Gengou proved

that there is absorption of the fixative substance by the bacilli of

swine erysipelas and of anthrax when placed in contact with the specific

serums of the immunised animals. In order that the protective power may

manifest itself adequately, therefore, besides the fixative substance,

some other factor capable of acting is also necessary.

[Sidenote: [284]]

In connection with my work on immunity against the micro-organism of

swine pneumo-enteritis I was able to demonstrate that the serum of

vaccinated rabbits, incapable of preventing the multiplication of the

specific cocco-bacillus, is also powerless to deprive it of its

virulence; it is without the power of causing its agglutination or of

neutralising its toxin. In short, this serum appears to exercise no

direct action on the micro-organism, yet, in spite of that, it prevents

its pathogenic action. With these results before me, I was led to assume

a certain stimulating action of the serum on the defensive elements of

the animal organism and especially on the phagocytic system. The

discovery of the fixative property of serums would lead us to believe

that this stimulation was entirely useless, and that the permeation of

micro-organisms by the fixative was amply sufficient to bring about

their destruction and removal from the animal. A living micro-organism

in its normal form, endowed with full virulence and provided with its

fighting weapon, the toxin, but at the same time permeated by the

fixative substance, might behave in the animal in some special way. It

might excite a strong positive chemiotaxis of the leucocytes and be

ingested and destroyed by these cells with greater facility. _A priori_,

there would be nothing to object to in this view, but certain facts are

opposed to it. Thus, in the case of micro-organisms just cited, we see

bacteria, permeated not only with the fixative but also with cytases,

capable of producing a fatal infection. We are thus compelled to accept

the theory of an influence of protective serums not only on the

micro-organisms but also on the organism of the animal into which they

are introduced. As this influence manifests itself in the form of a

strong phagocytosis, it is only natural that we should attribute it to

the existence of a _stimulating action_ of the serums of vaccinated

animals on the phagocytes of the normal animals. The detailed analysis

of the mechanism of the immunity acquired as the result of the injection

of these serums, as we shall attempt to prove in the following chapter,

in many cases confirms this view.

[Sidenote: [285]]

The important part played by the stimulation of the phagocytic reaction

in acquired immunity is supported by yet another series of facts and

from a different side. It has been clearly established that not only the

serum of immunised animals but also that of normal man and normal

animals, themselves susceptible to the pathogenic action of the

micro-organisms, protects the animal organism against infection. This

fact was first demonstrated in connection with researches on the

vaccination of guinea-pigs against the experimental peritonitis produced

by the cholera vibrio.

G. Klemperer[412] was the first to observe that the blood of several

individuals who had never had cholera was, nevertheless, in the case of

guinea-pigs, protective against peritoneal infection by the cholera

vibrio. He concluded therefrom that the individuals who had furnished

this protective blood possessed immunity against cholera. Soon

afterwards I[413] was able to extend analogous researches over a large

number of persons and to show that the protective power of the blood is

of very wide distribution in human beings. But, instead of assuming that

all these individuals, whose fluids protect the guinea-pig from

peritoneal infection, possess a natural immunity against cholera, I came

to the conclusion that the protective power of the blood cannot be taken

as a measure of the immunity of the individual from whom the blood was

drawn. Here again I assumed a stimulant action of the human blood on the

phagocytic reaction of the guinea-pig, looking upon it as quite natural

that the blood, capable of exciting the reaction in an alien animal,

might remain inactive in the body of the animal which furnished it.

[Sidenote: [286]]

R. Pfeiffer[414] has given much attention to the protective action of

serums; he has laid special stress on the essential difference between

the influence of normal serums and of those obtained from animals that

have acquired immunity. Whilst, in order to obtain a protective effect

with the normal blood or serum of man and animals, it is necessary to

inject a considerable quantity (from 0·5 c.c. upwards), the specific

serum, i.e. serum obtained from persons recovered from cholera or from

animals vaccinated against the cholera vibrio, is active in a very

minute dose. Sometimes the cholera peritonitis of the guinea-pig is

prevented by a fraction of a milligramme of such serum[415]. Based on

these facts, Pfeiffer has expressed the view that the normal serum acts

by stimulating the natural powers of defence of the animal, whilst the

specific serum exercises its influence in virtue of the property of

causing the formation of a special secretion which acts only against the

micro-organism which served for the production of the immunity. Pfeiffer

and his collaborators have demonstrated that normal serums are

protective, not only against the cholera vibrio, but also against

several other micro-organisms, e.g. the typhoid bacillus. One of his

pupils, Voges[416], believed that, in certain infections, the protective

power of normal blood might be greatly exaggerated, and that, in these

cases, the limit between the activity of normal and of specific serums

might be almost completely effaced. He affirmed, especially, that very

small doses (0·1 c.c.) of blood serum from a normal guinea-pig was quite

sufficient to prevent, in other guinea-pigs, a fatal infection by the

micro-organism of hog cholera and its allies. As this fact might be of

general application I asked M. Saltykoff[417], who was working in my

laboratory, to verify the statements of Voges. Several series of

experiments demonstrated the incorrectness of the contention. The small

doses of normal serum of guinea-pigs, indicated by Voges, were found to

be absolutely incapable of protecting against the virus used by him in

his experiments.

The fact that normal serums, injected in sufficiently large doses,

exhibited an undoubted protective property, affords additional proof

that this property cannot be identified with the fixative power. The

latter was present in serums which were not protective; here, then, we

have the inverse phenomenon and we see normal serums exercise their

protective action although they contain no fixative. This follows from

Bordet and Gengou’s experiments already described, according to which

the cytases, placed in contact with micro-organisms in normal serums,

remain free, simply because of the absence of fixatives.

We are led, then, from these demonstrations to recognise the presence of

stimulins not only in specific serums, but also in normal serums.

Between the two there is this difference that, when applied with the

normal fluids, the stimulins alone act, whilst when injected with the

serum of the animal enjoying acquired immunity the action of the

stimulins is facilitated and reinforced by the fixatives or sometimes,

perhaps, by the agglutinins.

[Sidenote: [287]]

[Sidenote: [288]]

The stimulating influence of certain normal serums may be so

considerable that it may prevent infection by the micro-organism,

injected at the same time in a dose many times more than lethal.

Wassermann[418] protected guinea-pigs by injecting into the peritoneal

cavity a quantity as great as 40 times the lethal dose of typhoid

bacilli, by introducing at the same time and at the same place 3 c.c. of

normal rabbit’s serum, heated to 60° C. Besredka[419], who confirmed

this observation, has analysed its special mechanism. He showed that the

serum exercises a very marked stimulating influence on the guinea-pig’s

leucocytes, which then exhibit a truly extraordinary phagocytic

activity. They are seen to act in the peritoneal fluid, but they are

much more active in the region of the omentum, where the leucocytes

gorge themselves with micro-organisms, devouring them by dozens. The

stimulating action of the heated rabbit’s serum is exercised in a

similar fashion if, instead of micro-organisms, grains of carmine be

injected. Very shortly after the commencement of the experiment very

little carmine is found outside the cells; it is all either ingested by

individual leucocytes, if the grains are small, or surrounded by

numerous leucocytes when the grains are massed together; this

phagocytosis is most developed in the region of the omentum, exactly as

in the case of typhoid bacilli.

These facts, which so clearly demonstrate the stimulating action of the

normal rabbit’s serum, prove in another way that the stimulin resists

heating to 60° C., and that, in this respect, it resembles the

agglutinins and fixatives. This may afford us an indication as to the

nature of the stimulating substance. The possibility of obtaining an

antistimulin gives us another valuable indication. Wassermann, in the

work we have just cited, showed that the serum of a rabbit, previously

treated with guinea-pig’s serum and injected under the same conditions

as in the experiment with normal rabbit’s serum, has completely lost its

protective power. The typhoid bacilli multiply freely in the peritoneal

cavity and the organism of the guinea-pig is incapable of opposing a

sufficient resistance. Wassermann thinks that, in this case, the disease

becomes grave because of the anticytase found in the serum of rabbits

treated with guinea-pig’s blood. There is no doubt that this serum is

really anticytasic. But as the free cytases found in the peritoneal

cavity of a guinea-pig inoculated at the moment of phagolysis, become

inactive under the influence of the anticytase and play merely a minor

part, it is impossible to accept the German investigator’s

interpretation. Indeed, Besredka has proved that, in this case, it is

the antiphagocytic or antistimulant action of the rabbit’s serum which

brings about the fatal issue in the case of the typhoid inoculation.

We have laid stress on the point that an animal, whose serum is

protective when introduced into another animal, may itself not be

refractory against the specific micro-organism. As regards the serum of

normal unvaccinated animals this has been so fully demonstrated that

nowadays no one doubts it. The question is more complicated in the case

of animals that have acquired immunity. As in the great majority of

cases the serum of these animals is found to be endowed with a very

great protective power, it has been accepted as proved that the animal

which furnishes it must itself possess great immunity. The degree of

protective power has even been taken as the measure of the acquired

immunity. Thus, the numerous attempts to vaccinate the human subject

against typhoid fever, undertaken in consequence of the researches of

Pfeiffer and Kolle[420], were based on the fact that in these cases the

serum of vaccinated individuals acquires a great protective power. It

was argued that if this power is present it can only be due to the

acquired immunity of the individuals who furnish such a serum.

Undoubtedly the protective property of the fluids and the resistance are

often equal; but it is none the less true that there are cases where, in

spite of this property being markedly developed, the animal that

furnishes the protective serum is susceptible to the action of the

micro-organism and may even succumb to infection therewith.

[Sidenote: [289]]

As the hypothesis just mentioned is of importance from a general point

of view it must be supported by adequate proof. It was during the course

of the vaccination of rabbits against the micro-organism of the

pneumo-enteritis epidemic at Gentilly that I was first able[421] to

assure myself of its accuracy. I noticed that some of these rabbits,

although vaccinated, ultimately succumbed to pyaemia, set up solely by

this micro-organism. They were consequently not refractory against the

disease, and yet their blood serum, when injected into normal rabbits

along with an absolutely fatal dose of micro-organisms, was found to be

highly protective. This observation drove me to the conclusion that the

protective power is not a function of immunity and cannot be received as

a measure of this immunity. Analogous facts have since been demonstrated

in certain other cases. Thus, Pfeiffer[422] on several occasions has

found that guinea-pigs, highly immunised against the cholera vibrio,

have succumbed after the injection of a moderate quantity of these

organisms. “On post-mortem examination of these cases living vibrios

were found in the peritoneal cavity, sometimes in considerable numbers;

and yet minimal doses of the heart blood given to normal guinea-pigs

caused in these animals a very marked breaking down of the vibrios.”

Alongside these facts may be placed others, described in the preceding

chapter, of well immunised animals dying from infection, after they had

been weakened by opium, cold, or other lowering agent. It is clearly

seen, then, that for the manifestation of acquired immunity it is

necessary that the reaction of the living cell elements should take

place without let or hindrance. When this reaction fails, the possession

of even great protective power is insufficient to prevent the immunised

animal from contracting a fatal infection.

[Sidenote: [290]]

[Sidenote: [291]]

If, in acquired immunity against micro-organisms, it is really the cell

defence which plays the most important part, we can readily imagine

cases where it by itself can confer immunity without calling in the

co-operation of the protective power of the fluids. When in this

connection we study the resistance of an animal against various

pathogenic organisms, we note, first of all, the very great variability

that exists in the production of the acquired humoral properties. In

certain cases, as in vaccination against vibrios or typhoid bacilli, the

serum very readily becomes not only protective, but agglutinative and

fixative. In other cases these properties develop with difficulty and

are only manifested after a long period of vaccination. Such is the case

with anthrax. After the discovery of protective serums, numerous

attempts were made to obtain a serum protective against the anthrax

bacillus. Several observers failed in their attempts, others were more

fortunate. Sclavo[423] and Marchoux[424] were the first to succeed in

obtaining a protective serum from animals hyperimmunised against

anthrax. They were able to show that the serum of sheep, treated first

with vaccines and then repeatedly with anthrax virus, would protect

rabbits against a fatal dose of the bacillus. Marchoux even obtained,

with hyperimmunised rabbits, a serum which prevented normal rabbits from

contracting fatal anthrax. Sobernheim[425] was less fortunate in his

first experiments. He satisfied himself that the blood serum of cattle

that had recovered spontaneously from anthrax or that had been

vaccinated according to Pasteur’s method, was absolutely unable to

protect small animals against the anthrax bacillus, and his

hypervaccinated rabbits furnished serums of doubtful activity. It was

only later that he succeeded[426] in obtaining better results;

especially when he used sheep. Even then he found that in the production

of the anti-infective property the individuality of the immunised

animals had a dominant influence. Thus, in two sheep, treated in exactly

the same way, the serum of one was found to be incapable of protecting a

rabbit, whilst that of the other exhibited an undoubted, although

feeble, protective power.

But what is of greater interest to us, from our point of view, is that

guinea-pigs which have been vaccinated against anthrax and which enjoy a

considerable immunity against this disease, exhibit no protective power.

In a letter from Behring I learnt that this fact had for the first time

been demonstrated by Wernicke in experiments carried out in the Hygienic

Institute at Marburg. After repeated and painstaking attempts this

observer succeeded in vaccinating guinea-pigs against enormous doses of

virulent anthrax bacilli. The serum from the animals so immunised was,

however, quite incapable of protecting normal guinea-pigs against a

fatal infection. This result was the more extraordinary since Wernicke’s

pigeons, likewise vaccinated against anthrax, gave a serum whose

protective power was quite distinct. Realising the great importance of

these facts I asked M. de Nittis[427] to repeat these experiments in my

laboratory. The vaccination of pigeons is an easy matter, but that of

guinea-pigs presents great difficulties. He succeeded, nevertheless, in

vaccinating some of these rodents very highly, and this enabled him to

compare the protective power of the blood serum in the two species. That

of the vaccinated pigeon was found to be endowed with this power and

protected guinea-pigs and mice against virulent anthrax. The serum of

the immunised guinea-pigs, on the contrary, exhibited no protective

property, just as in Wernicke’s experiments. The guinea-pigs and mice,

into which this serum was injected at the same time as the anthrax

bacilli, died even when attenuated anthrax was used. We have, then, in

this case, an example of acquired immunity, independent of any

protective power of the fluids of the body.

In the course of their researches on the bacillus isolated by R.

Pfeiffer from persons attacked by influenza, Delius and Kolle[428] tried

to vaccinate susceptible animals (guinea-pigs) against this minute

organism and to immunise animals naturally refractory (dog, sheep, goat)

against fairly large doses of cultures. They succeeded in vaccinating

guinea-pigs against ten times the lethal dose, but never obtained any

protective serum. Nor did the other animals that were treated furnish a

protective serum. “From the whole of our experiments carried on for

several years”—conclude Delius and Kolle—“it is quite evident that we

were unable to produce any appreciable change in the blood by the use of

those methods which have produced specific immunising serums against

other bacteria such as the bacilli of diphtheria, cholera, typhoid

fever, and ‘blue pus’” (p. 345). Slatineano undertook a detailed study

of Pfeiffer’s bacillus in my laboratory, but he found it impossible to

demonstrate any unquestionable protective effect exerted by the blood

serum of vaccinated guinea-pigs upon normal guinea-pigs inoculated with

a fatal dose of this organism. We are not justified, therefore, in

classing this bacillus with the anthrax bacillus; we may, however, cite

it as an argument illustrating the difficulty that is met with, in

certain examples of acquired immunity, of discovering the protective

power, when feeble and masked.

The inoculation with micro-organisms of animal nature causes the

development of acquired immunity, but in this case the properties of the

fluids of the body are but little in evidence or they may be even _nil_.

Let us return to the example of the _Trypanosoma_ of the rat which

excites in vaccinated animals a protective and weakly agglutinative

power of the serum. This fluid, however, is usually found to be

incapable even of rendering the flagellated parasites motionless.

[Sidenote: [292]]

The question of immunity against malaria has been much discussed. It is

well known that a first attack of this disease, so far from conferring

any immunity of the slightest durability, leaves a certain

predisposition to another attack. In spite of this the study of malaria

in various countries and in individuals belonging to different races has

demonstrated that there does indeed exist a certain degree of acquired

immunity against this disease. During recent years Koch[429] has paid

special attention to this subject and has furnished us with very

valuable data, based especially on a comparative study of the blood of

children and adults. The frequency of Laveran’s parasite in the former

and its rarity in the latter, have led him to the conclusion that

infantile malaria sets up an immunity which persists in the adult.

Moreover, it has been established that in malarial countries the

indigenous inhabitants exhibit an attenuated form of the disease,

unaccompanied by acute attacks, but with phenomena that are chronic and

very slow in development.

In spite of the existence of a certain degree of acquired immunity

against malaria, all attempts to demonstrate any protective action of

the serum have been fruitless. Celli[430], indeed, injected, as a

preventive, the blood serum of individuals who had recovered from

malaria or of others who were bled during the period of defervescence

following an acute crisis of this disease, but in every instance these

injections were found to be useless in preventing an attack of malaria.

We can readily understand that in a disease which is exclusively human,

such as malaria, it has not been possible to perform a sufficient number

of experiments to decide the question of the protective property of the

blood. In this respect we shall have greater chance of obtaining

satisfactory data if we direct our attention to some analogous disease

attacking one of the lower animals. Such a disease we have in Texas

fever, occurring in the Bovidae, as the result of the action of an

animal parasite, _Piroplasma bigeminum_, which invades the red blood

corpuscles much as Laveran’s parasite invades those of the human

subject.

[Sidenote: [293]]

[Sidenote: [294]]

As mentioned in the preceding chapter, Smith and Kilborne and Koch have

demonstrated that the Bovidae may acquire a real immunity against Texas

fever. Nicolle and Adil Bey[431] at Constantinople found indigenous

races that exhibited a remarkable immunity against the _Piroplasma_.

Having demonstrated this fact the idea occurred to them to inoculate

these refractory cattle with very large quantities of virulent blood and

to make use of the serum from animals so treated for the prevention of

infection in susceptible races of Bovidae. This experiment gave negative

results. Lignières[432] elaborated a special method of vaccinating

susceptible Bovidae and was successful in obtaining very encouraging

results. A commission of veterinary surgeons from Alfort[433] appointed

to verify these observations came to the conclusion that “the

vaccination as carried out by Lignières was absolutely effective.”

Lignières also carried out researches on the protective power of the

blood serum of his immunised cattle. In a communication to the

International Congress of Medicine, held in Paris in 1900, he stated

that the injection of several hundred cubic centimetres of this fluid

did not protect normal animals against infection. We must conclude,

therefore, that, here also, we have another example of acquired immunity

unaccompanied by the presence of any protective property of the blood

fluid.

These results have received confirmation from a most authoritative

source. Nocard has kindly communicated to me the fact that he has tried

in vain to confer immunity on normal dogs into which he has injected

blood serum coming from dogs that had recovered from the disease

produced by a haematozoon closely allied to that of Texas fever or serum

from sheep immunised with blood from the affected dogs.

Looking at the data we have just summarised as a whole, we are compelled

to recognise that, on the one hand, the protective power of the body

fluids may coincide with a susceptibility to the corresponding

micro-organism, and that, on the other, real acquired immunity may exist

without any manifestation of this humoral property, especially as, even

in immunised animals, the acquired immunity often persists longer than

does this property. It must be accepted then, that, in this immunity,

there exists something other than the powers of the fluids of the body,

that is to say, the factor which plays the predominant part is to be

sought for in the cellular elements. We need only recall the many facts

collected in the preceding chapter to be convinced that in acquired

immunity phagocytosis is the most constant and most general phenomenon.

We find it in cases where the humoral properties are the most marked, as

well as in those in which they are only slightly developed or are

entirely absent. We need not again discuss Pfeiffer’s phenomenon

analysed in the preceding chapter. It is sufficient to mention that this

example of the extracellular destruction of micro-organisms only occurs

under limited and special conditions. It is observed only in cases where

the injection is made into a situation rich in leucocytes which undergo

phagolysis as a result of the sudden change brought about in their

conditions of existence. Further, this phenomenon is observed only in

connection with micro-organisms that are slightly resistant to the

influence of the microcytases. In those cases in which we meet with

Pfeiffer’s phenomenon, we also meet with a widely extended phagocytic

reaction.

[Sidenote: [295]]

This reaction is most pronounced where the properties of the body fluids

are only slightly developed or are absent. The study of acquired

immunity against anthrax provides us with a very convincing proof of

this. We have already cited the example of vaccinated rabbits and rats

in which phagolysis is incomparably greater than in the susceptible

control animals which contract a fatal anthrax. This rule is general. It

is confirmed in the vaccinated sheep and guinea-pig. The absence, or

feeble development, of the protective power of the blood or of the other

humoral properties in no way, then, prevents the considerable change

which is set up in the phagocytes of animals that have acquired immunity

against anthrax. The negative chemiotaxis of the leucocytes, so marked

in susceptible animals, is modified into positive chemiotaxis as the

result of vaccination. This fact, one of fundamental importance, was

first demonstrated for the immunity against anthrax, later being

extended to other micro-organisms. Massart[434] studied the general

subject and collected a series of data which led him to say that

“vaccination effects an education of the leucocytes; these latter become

so adapted that they can approach the virulent micro-organisms.” The

best method of forming an estimate of the change which the leucocytes

undergo is by injecting subcutaneously very virulent micro-organisms

capable of setting up a generalised infection. The anthrax bacillus,

Gamaleia’s vibrio, the streptococci and the cocco-bacilli of swine and

fowl cholera are very suitable for such study. These micro-organisms,

when inoculated subcutaneously into susceptible animals, set up a very

slight local reaction or none at all, in the form of an exudation of

transparent fluid almost entirely without leucocytes. The

micro-organisms grow freely in these exudations and soon invade the

animal. In vaccinated animals the local reaction is more marked and the

exudation, very rich in leucocytes, is poor in fluid; the

micro-organisms remain free for a very short time, being soon ingested

by the leucocytes. Their destruction, inside these cells, takes a longer

or shorter time according to circumstances; but in the end it is always

complete.

[Sidenote: [296]]

The difference as regards phagocytic reaction between susceptible and

vaccinated animals, such as I have just described, has been generally

recognised by many observers. A few opponents are still found, however,

who consider that they are justified in affirming that the negative

chemiotaxis of the susceptible animal does not exist and that,

consequently, vaccination can in no way change it into positive

chemiotaxis. Werigo made himself the spokesman of this view, which he

has maintained in several papers[435]. Instead, however, of introducing

the virulent micro-organisms into the subcutaneous tissue of susceptible

animals he injected them directly into the veins. Using cultures of the

anthrax bacillus and of the cocco-bacillus of fowl cholera he injects

these into the venous system of normal rabbits. The animals soon die

from general infection. If, however, these animals are killed shortly

after inoculation, it is found on examination of sections that many of

the micro-organisms have been ingested by the leucocytes. Werigo

concludes from these facts that in the higher animals the chemiotaxis is

always positive; but that it ends in the destruction of the

micro-organisms in the vaccinated animals, never bringing about this

result in susceptible animals. Taking all the data on this question into

consideration, it is easy to convince oneself that this view cannot be

accepted as correct, for not only the definite phenomena observed below

the skin but also the no less demonstrative process appearing in the

peritoneal cavity prove most clearly the existence of this negative

chemiotaxis of the leucocytes. I need only recall Bordet’s experiment on

the fate of streptococci and _Proteus vulgaris_ when injected together

into the peritoneal cavity of guinea-pigs. Whilst the _Proteus_ bacilli

at the end of a very short time are all ingested by the leucocytes, the

streptococci remain free in the peritoneal fluid up to the death of the

animal. The leucocytes which exhibit a positive chemiotaxis as regards

the former, manifest a negative chemiotaxis as regards the streptococci.

[Sidenote: [297]]

In spite of the great force of these arguments, the discovery of a means

of reconciling the results obtained from the inoculation of

micro-organisms subcutaneously or into the peritoneal cavity, with those

observed after they had been injected into the blood vessels would be of

great interest, and Zilberberg and Zeliony[436] have undertaken a series

of experiments with this object. Following Werigo they made use of the

cocco-bacilli of fowl cholera, and found, in accordance with his

observations, that the intravenous injection of these organisms,

obtained from cultures in nutrient media, causes a very marked

phagocytosis of the cocco-bacilli. When, however, they injected into the

veins of rabbits cocco-bacilli that had been grown in the peritoneal

fluid of other rabbits, they found the micro-organisms free in the blood

plasma and observed only a very restricted phagocytosis in the

microphages of the liver. It follows from these experiments that the

ingestion of the cocco-bacilli, in Werigo’s experiments, was dependent

on the presence of a large number of attenuated micro-organisms which

were present in the cultures that he employed for his injections.

Alongside these organisms, slightly or not virulent, were others,

endowed with their normal pathogenic activity and quite numerous enough

to set up a fatal infection. When Zilberberg and Zeliony replaced

cultures on agar by the peritoneal exudation which contained virulent

cocco-bacilli almost exclusively, the phagocytosis in rabbits, injected

into the veins, was found to be almost suppressed. With the object of

establishing whether the absence of the phagocytic reaction, in this

case, really depended on negative chemiotaxis on the part of the

leucocytes, the above cited observers performed the following

experiment. They injected into the vein of a rabbit, already affected

with a generalised infection by the cocco-bacillus of fowl cholera, an

innocuous culture of a saprophytic staphylococcus. Post-mortem

examination showed that these cocci were almost entirely ingested by the

same phagocytes which refused so energetically to seize the

cocco-bacilli. This experiment, analogous to that of Bordet on

streptococcus and _Proteus_, compels us to reject Werigo’s conclusions

as to the absence of negative chemiotaxis in the phagocytes of the

higher animals. I ought to add that the work of Zilberberg and Zeliony

was in part executed in my laboratory so that I was able to convince

myself by ocular demonstration of the complete accuracy of their

statements.

Independently of these observers and even before their work appeared,

Th. Tchistovitch[437] published an interesting study on the same

question. He injected very virulent streptococci into the ear vein of

rabbits. These micro-organisms set up a generalised and fatal infection

in which phagocytosis was completely absent or nearly so. Here again was

manifested a negative chemiotaxis of the phagocytes, which, henceforth,

could no longer be questioned.

[Sidenote: [298]]

In certain infective diseases terminating fatally a very marked

phagocytosis is observed even in susceptible animals. The most typical

example of this is furnished by swine erysipelas and mouse septicaemia.

We know from the researches of Koch[438], followed by those of

Loeffler[439], Schütz[440] and others, that in animals which have died

from these two diseases the leucocytes are gorged with small specific

bacilli. A method of vaccinating animals against the micro-organism of

swine erysipelas was worked out by Pasteur and Thuillier[441] and was

afterwards studied by many observers. Thanks to this method it has been

possible to demonstrate the phenomena which may be observed in

vaccinated animals (especially rabbits). Here also a phagocytosis takes

place, even more rapid and more complete than in susceptible animals.

What is more important, the intracellular digestion of the ingested

bacilli is followed by the total destruction of the micro-organisms in

the vaccinated animals, though in the normal animals this digestion is

very imperfect.

[Sidenote: [299]]

The acquisition of immunity against micro-organisms is, therefore, due

not only to the change from negative to positive chemiotaxis, but also

to the perfecting of the phagocytic and digestive powers of the

leucocytes—a general superactivity and adaptation of the phagocytic

reaction of the immunised animal is produced. This conclusion, based

upon a large number of well-established facts and in complete harmony

with the whole of the data at our disposal concerning acquired immunity,

has been attacked by Denys and Leclef[442] in their work on the

streptococcus. They base their opposition upon experiments made _in

vitro_ on the action of serums and leucocytes on this micro-organism.

They have compared the bactericidal power of mixtures of the serums of

normal and of vaccinated rabbits with leucocytes isolated from

exudations from these two groups of animals. The leucocytes, whether

derived from normal or from vaccinated rabbits, when mixed with normal

serum were equally incapable of ingesting and destroying the

streptococci. When mixed with blood serum from vaccinated rabbits,

however, the two kinds of leucocytes exhibited a very marked phagocytic

reaction. Denys and Leclef conclude from this that phagocytosis,

although an important factor in immunity, plays merely a secondary part

and is dependent on the humoral properties. The experiments and views of

these two observers have been generally received by the partisans of the

bactericidal theory of the body fluids as an actual proof of this

theory. We cannot agree. Researches extending over a long period have

shown us that the study of phagocytosis _in vitro_ can give only a very

inexact and imperfect idea of the course of the phenomena in the living

animal. Usually the leucocytes taken from the exudations, although

amoeboid, no longer fulfil their phagocytic functions at a time when in

the animal they would ingest micro-organisms with the greatest rapidity.

As a general rule, existence outside the living body weakens them very

considerably. But in some cases, rare it is true, the leucocytes

although inactive in the animal exhibit intense phagocytosis when

introduced into a hanging drop of fluid from an exudation or even of

urine. In any case it is very hazardous to infer from phenomena which

appear under these artificial conditions what takes place in the living

animal. The value of the experiments of Denys and Leclef is still

further marred by the fact that they mixed the leucocytes with blood

serum. They appear to have lost sight of the fact that this fluid is far

from corresponding to that which bathes the leucocytes in the living

animal. The serums contain leucotoxin in greater or less quantity and it

is not to be wondered at that the leucocytes when mixed with normal

rabbit’s serum should perish very rapidly. Further, the serum of

vaccinated rabbits is agglutinative (this fact, however, was not

sufficiently elucidated in 1894 when the researches of Denys and Leclef

were made) and the clumping of streptococci might simulate their

destruction. In a word, the experiments of these observers have been

carried out under such conditions that it is impossible to base upon

them a refutation of data obtained in the living animal. Moreover, in

the description of the phenomena which appear in the subcutaneous tissue

of rabbits inoculated with the streptococcus, Denys and Leclef provide

us with arguments against their own view.

[Sidenote: [300]]

These observers introduce the same quantity of streptococci below the

skin of the ear of normal and of vaccinated rabbits. In the first there

is soon produced a very marked oedema of the ear, in which may be seen a

number of streptococci and of leucocytes that have not ingested any

micro-organisms. In the second the oedema does not develop, but at the

seat of invasion a number of leucocytes come up and these soon ingest

the streptococci. As we see, the phenomena manifest themselves here just

as they do with the anthrax bacillus and many other micro-organisms when

under analogous conditions. Denys and Leclef, indeed, recognise that,

below the skin of the ear of vaccinated rabbits, the small quantity of

exudation fluid is not sufficient to enable us to accept it as capable

of exerting any considerable influence as regards humoral properties.

Nevertheless, they think that the “serum” of this fluid may exercise a

certain action, but they furnish no proof of this, and seem to ignore

the fact that the plasma of the subcutaneous exudation is far from being

identical with blood serum obtained outside the animal. At present it is

well known that this latter fluid contains cytases which are absent from

the plasmas. Now, the feeble bactericidal action, if this really exists

as regards the streptococcus, must be attributed to the microcytase

which has escaped from the leucocytes at the time of the preparation of

the serum.

To sum up, the example studied by Denys and Leclef clearly comes under

the general law of phagocytic reaction in acquired immunity against

micro-organisms. It is impossible to deny that the superactivity of the

phagocytes which is always found in this immunity, although readily

observed, cannot be demonstrated in a rigorous fashion outside the

fluids which bathe the cells. There are, however, very important

analogies which may be invoked in favour of this thesis. We have already

cited in our fifth chapter Delezenne’s experiments on the digestion of

gelatine by the leucocytes of the dog, which show in the most

demonstrative fashion that these cells accustom themselves to bring

about this digestion more and more quickly and this quite independently

of any humoral influence.

For some time past there has been no doubt as to the fundamental fact

that the phagocytes in immunised animals seize and destroy living

micro-organisms. Several attempts have been made to show that such

destruction of these bacteria takes place solely by the body fluids, and

that the phagocytes intervene only as “scavengers” to carry off the dead

bodies of the micro-organisms. The numerous observations, described in

the preceding chapter, absolve us from again entering into a discussion

of this question. Moreover, the majority of these opponents now

recognise that micro-organisms are ingested in a living state by the

phagocytes of immunised animals. Some, however, have expressed the

opinion that these living micro-organisms, before becoming the prey of

the phagocytes, must undergo some preliminary attenuation of virulence

through the action of the body fluids. Hence the theory of the

attenuating power of the fluids of the body, maintained especially by

Bouchard and his pupils. During the course of our exposition of the

facts concerning acquired immunity, we have several times had occasion

to speak of the virulence of micro-organisms in the immunised animal.

Here, therefore, we may confine ourselves to a brief summary of the

observations collected on this point.

[Sidenote: [301]]

Having observed that the anthrax bacillus, when developed in the blood

of immunised sheep, was incapable of giving fatal anthrax to rabbits, I

expressed[443] the opinion that under these conditions its virulence had

become attenuated. Later, analogous changes were shown by Charrin[444]

in the _Bacillus pyocyaneus_ when cultivated in the serum of immunised

animals. Bouchard[445], generalising on these data, arrived at the

following theory of vaccination. “The inoculation of a strong virus into

a vaccinated animal is equivalent to the inoculation of an attenuated

virus. The attenuation, however, instead of being done beforehand in the

laboratory, is brought about in the tissues of the vaccinated animal”

(p. 18). Charrin and Roger[446] upheld this view, and the latter offered

several new arguments in support of it. He observed that animals

inoculated with pneumococci and streptococci grown in the blood serum of

vaccinated animals, contracted a transient and benign disease merely,

whilst the control animals, inoculated with the same micro-organisms,

cultivated in normal serum, always died from generalised infection.

[Sidenote: [302]]

The discovery of the protective property of serums has thrown a new

light upon these experiments. We must now ask ourselves: Does the

innocuousness of micro-organisms depend not on the attenuation of the

virus, but rather on the protective action of the serum itself? When, in

the course of my researches on the Gentilly cocco-bacillus, I found that

this organism, cultivated in the serum of vaccinated rabbits, became

much less pathogenic than when it was grown in the serum of normal

rabbits, I set myself to answer this question. Simple filtration through

paper was sufficient to rid the organism of the serum in which it had

grown. The inoculation of cocco-bacilli thus treated proved at once that

their virulence was in no degree modified, and that it was the

intervention of the serum that prevented the micro-organism from setting

up the rapidly fatal disease. Issaeff[447], who, in my laboratory,

carried out the investigation, was able to extend this to the

pneumococcus. He obtained agglutinated cultures in the serum of

vaccinated rabbits, and he compared their activity by injecting them (1)

with, and (2) without their culture medium. The difference was very

marked. In the first case the infection produced was much slower in its

course than in the second. The virulence of the washed pneumococci was

found to be the same whether they came from a culture in normal serum or

from one in immunised serum. Sanarelli[448] obtained the same result

with Gamaleia’s vibrio. The vibrios when grown in the serum of

vaccinated guinea-pigs proved to be very virulent so soon as they were

freed from the fluid in which they were grown. Later, similar

demonstrations were given by Bordet[449] and Mesnil[450] with respect to

streptococci and to the bacilli of swine erysipelas. We must, then,

conclude that we have here to do with a general law. Some experiments

made by de Nittis[451] might seem to indicate an exception to such a

law. He observed that anthrax bacilli when grown in the serum of

vaccinated pigeons lost a part of their virulence. It must not be

forgotten, however, that he grew his cultures under special conditions;

the bacillus was grown for several days at 42° C., this in itself being

quite sufficient to bring about a certain attenuation of virulence.

The theory of the attenuating action of the body fluids, based on the

attenuation of the virus in the serum of vaccinated animals, can no

longer be maintained, as it is a well-established fact that the serum,

obtained outside the body, is a fluid differing in character and

properties from the plasma of the living animal. We have seen up to what

point this demonstration has shaken the theory of the bactericidal

action of the body fluids.

[Sidenote: [303]]

It cannot be doubted that a micro-organism may undergo a certain

weakening in virulence, as well as in certain other functions, in the

body of the animal that has acquired immunity. But the question must be

put: Is this effect obtained as the result of humoral or of cellular

action? As a general rule, exudations obtained from vaccinated animals,

and containing living micro-organisms, are found to be virulent when

inoculated directly into susceptible animals. This fact was established

by Pasteur[452] when he first carried out his researches on acquired

immunity against fowl cholera. He showed that the exudations of

vaccinated fowls set up a fatal disease in normal fowls, without there

being the least evidence of any attenuation of the micro-organism. The

same applies to the Gentilly cocco-bacillus and to the anthrax bacillus

in a very great majority of examples. De Nittis observed that the

exudations of immunised pigeons produced a fatal infection in the

guinea-pig and in the mouse. In the immunised guinea-pig, on the other

hand, he found that the exudations soon became innocuous for these

animals. This alteration, however, must be attributed not to the body

fluids (which exhibit no protective or attenuating power) but to the

action of the cells.

With the object of gaining some idea of the changes that the

micro-organisms undergo in the immunised animal, Vallée[453] carried out

a series of experiments on rabbits vaccinated against the bacillus of

swine erysipelas. He enclosed these bacilli in sacs of collodion which

he introduced into the peritoneal cavity of susceptible rabbits and of

others that were hyperimmunised. The bacillus developed well in both

cases. It gave homogeneous non-agglutinated cultures in the sacs placed

in normal animals, whilst in the sacs introduced into the peritoneal

cavity of hyperimmunised rabbits the bacilli grew into agglutinated

filaments. This proves that the wall of the sacs permitted of the

passage of the active substances elaborated in the immunised animal.

Different from the point of view of agglutination, the cultures likewise

exhibited a considerable difference in their pathogenic activity. The

cultures developed in the sacs in hyperimmunised rabbits were found to

be much more virulent than those grown in the sacs in control animals.

This augmentation of virulence depends, probably, on the influence of

the active substances which pass through the walls of the sacs. In any

case, this experiment affords further confirmation of the impossibility

of maintaining the theory of the attenuation of micro-organisms by the

fluids of an animal enjoying acquired immunity.

[Sidenote: [304]]

Since the discovery of the antitoxic property of the fluids of the body,

it has been accepted that its manifestation was indispensable for the

acquisition of immunity. It was thought that in order to get rid of

pathogenic micro-organisms the animal had first to develop the means of

neutralising their toxins. These substances once prevented from exerting

their toxic action, the micro-organisms were left without their weapon

of attack and found themselves reduced to the condition of simple

saprophytes. It was accepted, therefore, that an effective antitoxic

power was always to be found in the fluids of animals that had acquired

immunity. Against this explanation, however, are certain established

facts. Chauveau[454] had observed that Algerian sheep, whose natural

immunity was further strengthened by considerable doses of anthrax

bacilli, exhibited a susceptibility to injections of anthrax blood quite

as marked as that of normal sheep. The immunity against the virus, then,

did not progress _pari passu_ with that against the poison. Later,

Charrin and Gamaleia[455] furnished important data on this subject. They

showed that animals vaccinated against the _Bacillus pyocyaneus_ and the

vibrios of Koch and Gamaleia were even more susceptible to intoxication

by the soluble products of these micro-organisms than were normal

animals which had acquired no immunity against the corresponding

bacteria. Shortly afterwards this observation was confirmed by

Selander[456], in his work on hog cholera, carried out under Roux’s

direction. Rabbits vaccinated against the cocco-bacillus of this disease

resisted infection by the virus, but died as a result of the exhibition

of the same doses of toxin that killed normal rabbits. I[457] was able

not only to verify this, but to add to it the further fact that the

blood serum of vaccinated rabbits, although markedly protective against

infection, exercised not the slightest antitoxic action.

When, later, R. Pfeiffer set himself to study the immunity of animals

against the cholera vibrio, he, along with his collaborators, was able

to furnish numerous data confirming the hypothesis that animals

thoroughly vaccinated against this vibrio had not thereby become more

resistant to its toxin and that their anti-infective serum exhibited no

antitoxic power. These results have been confirmed repeatedly and must

be regarded as fully established.

[Sidenote: [305]]

Von Behring here recognised a general law which, with the aid of his

collaborators, he attempted to develop. We owe to him the knowledge that

the susceptibility, augmented as regards the toxins, of animals

vaccinated against micro-organisms, might even serve in doubtful cases

to reveal the presence of their bacterial poisons. Culture products when

deprived of micro-organisms often set up no poisoning in normal animals

susceptible to infection. From this fact it is generally concluded that

the toxin is not present in the products in question. But animals of the

same species when immunised against infection by the micro-organism,

owing to their “hypersusceptibility,” react much more delicately and

allow of the demonstration of the presence of bacterial poisons in

fluids inactive for unvaccinated animals.

In collaboration with Kitashima[458], von Behring immunised guinea-pigs

against the diphtheria bacillus, and demonstrated that two or three

injections of diphtheria toxin were quite sufficient to render these

animals refractory to infection by the diphtheria bacillus though they

became more susceptible to intoxication. Von Behring considers that this

augmentation of susceptibility to the diphtheria poison may be a means

of rendering the local reaction of the living elements at the point of

introduction of the bacilli more active.

In any case, it is beyond question that acquired immunity against

microbial infection is quite independent of the resistance against the

toxins of the corresponding micro-organism. An antitoxic manifestation

of any kind, therefore, cannot be regarded as necessary for the

development of immunity against the micro-organism.

Of all the humoral properties developed in acquired immunity against

micro-organisms, the fixative power and the protective power are the

most constant. It might naturally be suggested, as a result of this

observation, that these two powers are indispensable for the

manifestation of phagocytosis for the purpose of destroying and of

ridding the animal of the pathogenic organisms. It is quite possible to

understand how, under these conditions, the idea has been put forward

that anti-infective acquired immunity is the result of two different

factors: in the first place, a humoral property independent of the

phagocytes and, in the second place, the phagocytes themselves. But the

part played by these cells cannot be accepted as purely secondary—a view

which has been advanced and defended again and again. This question is

of such importance that it is reasonable to ask whence come the humoral

properties, such as the fixative power and the protective power, factors

of such far-reaching influence in anti-infective immunity?

[Sidenote: [306]]